TW202334189A - Tcr and peptides - Google Patents

Abstract

A T-cell receptor (TCR), which binds to a Wilms tumour 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC).

Description

TCR and peptides

The present invention relates to T cell receptors (TCRs) that bind to peptides derived from the Wilms tumor 1 protein (WT1) when presented via the major histocompatibility complex. In this regard, the present invention relates to complementarity determining regions (CDRs) that specifically recognize the WT1 peptide. The invention further relates to immunogenic peptides derived from WT1.

The basis of T cell receptor (TCR) gene therapy is the gene transfer of high-affinity tumor-specific TCR genes to T lymphocytes, which can specifically target the desired tumor-associated antigens and lead to less toxicity and more specific and effective therapy. This approach has shown promise in clinical trials. One of the main obstacles limiting the use of TCR gene therapy in the clinical treatment of cancer is the lack of tumor-specific T cells and corresponding TCRs. Therefore, the low availability of tumor-specific TCRs remains an open issue, which limits the widespread application of TCR-based immunotherapy approaches.

Most tumor-associated antigens (TAAs) are autologous and therefore T cells specific for these molecules are either destroyed or anergic due to central and peripheral tolerance. Nonetheless, after allogeneic hematopoietic stem cell transplantation (allo-HSCT), natural tumor-specific T cells can still be observed in healthy donors and patients, especially in patients affected by hematological malignancies. Lymphocyte frequency correlates with disease regression (Kapp, M. et al. Bone Marrow Transplantation 43, 399-410 (2009); and Tyler, E.M. et al. Blood 121, 308-317 (2013)).

The selection of tumor antigens to target with immunotherapeutic approaches remains a matter of debate. Ideal TAA has high expression on tumor cells and lowest expression in healthy tissues.

Wilms' tumor 1 (WT1) is an intracellular protein encoding a zinc-finger transcription factor that plays an important role in cell growth and differentiation (Yang, L. et al. Leukemia 21, 868-876 (2007)). WT1 is widely expressed in various blood and solid tumors, but shows limited expression in various healthy tissues (such as gonads, uterus, kidney, mesothelium, and progenitor cells in different tissues). Recent evidence suggests a role for WT1 in leukemogenesis and tumorigenesis.

Several ongoing clinical trials rely on the generation of cytotoxic T lymphocyte (CTL) responses following vaccination with the WT1 peptide. However, despite the recognition that WT1 can be used in immunotherapy, currently only a small number of WT1 epitopes from a limited number of HLA alleles are available for vaccination purposes (Di Stasi, A. et al., Front. Immunol. (2015) ). One such epitope is the WT1 126-134 epitope (RMFPNAPYL; SEQ ID NO: 71), which is presented by the MHC encoded by the HLA-A*0201 allele (ie, the epitope is restricted by HLA-A*0201).

Since the major histocompatibility complex (MHC) with the HLA-A*0201 haplotype is expressed in the vast majority (60%) of the Caucasian population, the HLA-A*0201-restricted epitope and corresponding TCR are of interest. Interest. Therefore, immunotherapy utilizing TCRs targeting HLA-A*0201-restricted WT1 epitopes is particularly advantageous as these TCRs are widely available.

The WT1 126-134 epitope has been studied extensively in several trials alone or in combination with complete tumor antigens. However, recent reports highlight major issues regarding the handling of this specific epitope, which may impair its use for immunotherapy purposes. Notably, the WT1 126-134 epitope is processed more efficiently by the immunoproteasome compared to the standard proteasome (Jaigirdar, A. et al. J Immunother. 39(3):105-16 (2016)). This results in poor recognition of many HLA-A*0201 tumor cell lines or primary leukemia cells that express WT1 endogenously.

Therefore, there is still a need for new WT1 epitopes, especially those presented by MHC with prevalent HLA haplotypes (eg, HLA-A*0201).

One naturally processed HLA-A*0201 restricted epitope that has been identified is WT1 37-45, which has the amino acid sequence VLDFAPPGA (SEQ ID NO: 72, see, e.g., Smithgall et al. 2001; Blood 98 (11, part 1 ): 121a). However, few TCR amino acid sequences, especially CDR sequences, specific for this peptide sequence have been reported (Schmitt, T.M. et al. (2017) Nat Biotechnol 35: 1188-1195).

Therefore, there is still a need for new WT1 epitopes, especially those limited to common HLA alleles, and new TCRs capable of binding to WT1 epitopes.

Novel TCRs have been identified that bind to WT1 peptide when presented via MHC. In addition, the amino acid sequence of the TCR, including that of its CDR region, has been determined, which is responsible for the binding specificity for WT1. Furthermore, T cells expressing TCRs according to the invention have been shown to specifically target and kill cells expressing WT1 protein. In addition, it has been shown that the TCR of the present invention is limited to MHC encoding HLA class I and class II alleles common in the Caucasian population, such as HLA-A*0201, HLA-B*38:01, HLA-C*03:03 or HLA-C*07:02.

In one aspect, the invention provides a T cell receptor (TCR) that binds to Wilms' tumor 1 protein (WT1) peptide when presented via the major histocompatibility complex (MHC), wherein the TCR: (i) Comprises CDR3α, which contains the amino acid sequence of CASGGGADGLTF (SEQ ID NO: 25) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CASGRGDTEAFF (SEQ ID NO: The amino acid sequence of 30) or its variant with up to three amino acid substitutions, additions or deletions; (ii) Comprises CDR3α, which contains the amino acid sequence of CAMRTGGGADGLTF (SEQ ID NO: 3) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CASSEAGLSYEQYF (SEQ ID NO: 8) The amino acid sequence or its variant with up to three amino acid substitutions, additions or deletions; (iii) Comprising CDR3α, which contains the amino acid sequence of CILSTRVWAGSYQLTF (SEQ ID NO: 14) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CATGQATQETQYF (SEQ ID NO: The amino acid sequence of 19) or its variant with up to three amino acid substitutions, additions or deletions; (iv) Comprises CDR3α, which contains the amino acid sequence of CAVIGGTDSWGKLQF (SEQ ID NO: 36) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CASSQEEGAVYGYTF (SEQ ID NO: The amino acid sequence of 41) or its variant with up to three amino acid substitutions, additions or deletions; (v) Comprises CDR3α, which contains the amino acid sequence of CAVIGGTDSWGKLQF (SEQ ID NO: 36) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CATSREGLAADTQYF (SEQ ID NO: The amino acid sequence of 52) or its variant with up to three amino acid substitutions, additions or deletions; (vi) Comprises CDR3α, which contains the amino acid sequence of CVVPRGLSTDSWGKLQF (SEQ ID NO: 47) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CATSREGLAADTQYF (SEQ ID NO: The amino acid sequence of 52) or its variant with up to three amino acid substitutions, additions or deletions; (vii) Comprise CDR3α, which comprises the amino acid sequence of CVVPRGLSTDSWGKLQF (SEQ ID NO: 47) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSQEEGAVYGYTF (SEQ ID NO: The amino acid sequence of 41) or its variant with up to three amino acid substitutions, additions or deletions; (viii) Comprising CDR3α, which comprises the amino acid sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSSGLAFYEQYF (SEQ ID NO: The amino acid sequence of 98) or its variants with up to three amino acid substitutions, additions or deletions; (ix) Comprises CDR3α, which contains the amino acid sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CASSQLSGRDSYEQYF (SEQ ID NO: The amino acid sequence of 104) or its variant with up to three amino acid substitutions, additions or deletions; (x) Comprises CDR3α, which contains the amino acid sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CASSTLGGELFF (SEQ ID NO: The amino acid sequence of 120) or its variant with up to three amino acid substitutions, additions or deletions; (xi) Comprises CDR3α, which contains the amino acid sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CASSTLGGELFF (SEQ ID NO: The amino acid sequence of 120) or its variant with up to three amino acid substitutions, additions or deletions; (xii) comprising CDR3α, which comprises the amino acid sequence of CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSLEGRAMPRDSHQETQYF (SEQ ID NO: The amino acid sequence of 136) or its variants with up to three amino acid substitutions, additions or deletions; (xiii) comprising CDR3α, which comprises the amino acid sequence of CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CATSWGLNEQYF (SEQ ID NO: The amino acid sequence of 142) or its variants with up to three amino acid substitutions, additions or deletions; (xiv) comprising CDR3α, which comprises the amino acid sequence of CAATSRDDMRF (SEQ ID NO: 131) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSLEGRAMPRDSHQETQYF (SEQ ID NO: The amino acid sequence of 136) or its variants with up to three amino acid substitutions, additions or deletions; (xv) Comprises CDR3α, which comprises the amino acid sequence of CAATSRDDMRF (SEQ ID NO: 131) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CATSWGLNEQYF (SEQ ID NO: The amino acid sequence of 142) or its variants with up to three amino acid substitutions, additions or deletions; (xvi) Comprising CDR3α, which comprises the amino acid sequence of CALPDKVIF (SEQ ID NO: 148) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSVSAGSTGELFF (SEQ ID NO: The amino acid sequence of 158) or its variant with up to three amino acid substitutions, additions or deletions; (xvii) Comprises CDR3α, which contains the amino acid sequence of CAGLYATNKLIF (SEQ ID NO: 153) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CASSVSAGSTGELFF (SEQ ID NO: The amino acid sequence of 158) or its variant with up to three amino acid substitutions, additions or deletions; (xviii) Comprises CDR3α, which contains the amino acid sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CASSTLGGELFF (SEQ ID NO: The amino acid sequence of 120) or its variant with up to three amino acid substitutions, additions or deletions; (xix) comprising CDR3α, which comprises the amino acid sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSSGLAFYEQYF (SEQ ID NO: The amino acid sequence of 98) or its variants with up to three amino acid substitutions, additions or deletions; (xx) Comprises CDR3α, which contains the amino acid sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CASSQLSGRDSYEQYF (SEQ ID NO: The amino acid sequence of 104) or its variant with up to three amino acid substitutions, additions or deletions; (xxi) Comprising CDR3α, which comprises the amino acid sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSSGLAFYEQYF (SEQ ID NO: 98) or a variant thereof having up to three amino acid substitutions, additions or deletions; or (xxii) Comprises CDR3α, which contains the amino acid sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which contains CASSQLSGRDSYEQYF (SEQ ID NO: The amino acid sequence of 104) or its variant with up to three amino acid substitutions, additions or deletions.

In one embodiment, the TCR contains the following CDR sequence: (i) CDR1α-NSAFQY (SEQ ID NO: 23), CDR2α-TYSSGN (SEQ ID NO: 24), CDR3α - CASGGGADGLTF (SEQ ID NO: 25), CDR1β-SGDLS (SEQ ID NO: 28), CDR2β-YYNGEE (SEQ ID NO: 29), and CDR3β-CASGRGDTEAFF (SEQ ID NO: 30), or variants thereof each having up to three amino acid substitutions, additions or deletions; (ii) CDR1α-TSDQSYG (SEQ ID NO: 1), CDR2α - QGSYDEQN (SEQ ID NO: 2), CDR3α - CAMRTGGGADGLTF (SEQ ID NO: 3), CDR1β-SNHLY (SEQ ID NO: 6), CDR2β - FYNNEI (SEQ ID NO: 7), and CDR3β - CASSEAGLSYEQYF (SEQ ID NO: 8), or variants thereof each having up to three amino acid substitutions, additions or deletions; (iii) CDR1α-TISGTDY (SEQ ID NO: 12), CDR2α-GLTSN (SEQ ID NO: 13), CDR3α - CILSTRVWAGSYQLTF (SEQ ID NO: 14), CDR1β-KGHDR (SEQ ID NO: 17), CDR2β-SFDVKD (SEQ ID NO: 18), and CDR3β-CATGQATQETQYF (SEQ ID NO: 19), or variants thereof each having up to three amino acid substitutions, additions or deletions; (iv) CDR1α-DRGSQS (SEQ ID NO: 34), CDR2α-IYSNGD (SEQ ID NO: 35), CDR3α - CAVIGGTDSWGKLQF (SEQ ID NO: 36), CDR1β - LGHNA (SEQ ID NO: 39), CDR2β-YSLEER (SEQ ID NO: 40), and CDR3β - CASSQEEGAVYGYTF (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions or deletions; (v) CDR1α-DRGSQS (SEQ ID NO: 34), CDR2α-IYSNGD (SEQ ID NO: 35), CDR3α - CAVIGGTDSWGKLQF (SEQ ID NO: 36), CDR1β-LNHNV (SEQ ID NO: 50), CDR2β - YYDKDF (SEQ ID NO: 51), and CDR3β - CATSREGLAADTQYF (SEQ ID NO: 52), or variants thereof each having up to three amino acid substitutions, additions or deletions; (vi) CDR1α-NSASQS (SEQ ID NO: 45), CDR2α - VYSSGN (SEQ ID NO: 46), CDR3α - CVVPRGLSTDSWGKLQF (SEQ ID NO: 47), CDR1β-LNHNV (SEQ ID NO: 50), CDR2β - YYDKDF (SEQ ID NO: 51), and CDR3β - CATSREGLAADTQYF (SEQ ID NO: 52), or variants thereof each having up to three amino acid substitutions, additions or deletions; (vii) CDR1α-NSASQS (SEQ ID NO: 45), CDR2α - VYSSGN (SEQ ID NO: 46), CDR3α - CVVPRGLSTDSWGKLQF (SEQ ID NO: 47), CDR1β - LGHNA (SEQ ID NO: 39), CDR2β-YSLEER (SEQ ID NO: 40), and CDR3β - CASSQEEGAVYGYTF (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions or deletions; (viii) CDR1α - VSNAYN (SEQ ID NO: 91), CDR2α-GSKP (SEQ ID NO: 92), CDR3α-CAAPNDYKLSF (SEQ ID NO: 93), CDR1β-SEHNR (SEQ ID NO: 96), CDR2β - FQNEAQ (SEQ ID NO: 97), and CDR3β - CASSSGLAFYEQYF (SEQ ID NO: 98), or variants thereof each having up to three amino acid substitutions, additions or deletions; (ix) CDR1α-VSNAYN (SEQ ID NO: 91), CDR2α-GSKP (SEQ ID NO: 92), CDR3α-CAAPNDYKLSF (SEQ ID NO: 93), CDR1β-SGHDN (SEQ ID NO: 102), CDR2β-FVKESK (SEQ ID NO: 103), and CDR3β - CASSQLSGRDSYEQYF (SEQ ID NO: 104), or variants thereof each having up to three amino acid substitutions, additions or deletions; (x) CDR1α-VSGNPY (SEQ ID NO: 108), CDR2α - YITGDNLV (SEQ ID NO: 109), CDR3α - CAVRDGGATNKLIF (SEQ ID NO: 110), CDR1β-MNHEY (SEQ ID NO: 118), CDR2β-SMNVEV (SEQ ID NO: 119), and CDR3β-CASSTLGGELFF (SEQ ID NO: 120), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xi) CDR1α-NIATNDY (SEQ ID NO: 113), CDR2α - GYKTK (SEQ ID NO: 114), CDR3α - CLVGGYTGGFKTIF (SEQ ID NO: 115), CDR1β-MNHEY (SEQ ID NO: 118), CDR2β-SMNVEV (SEQ ID NO: 119), and CDR3β-CASSTLGGELFF (SEQ ID NO: 120), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xii) CDR1α-SSVSVY (SEQ ID NO: 124), CDR2α-YLSGSTLV (SEQ ID NO: 125), CDR3α - CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126), CDR1β-SEHNR (SEQ ID NO: 134), CDR2β - FQNEAQ (SEQ ID NO: 135), and CDR3β - CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xiii) CDR1α-SSVSVY (SEQ ID NO: 124), CDR2α-YLSGSTLV (SEQ ID NO: 125), CDR3α - CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126), CDR1β-LNHNV (SEQ ID NO: 140), CDR2β - YYDKDF (SEQ ID NO: 141), and CDR3β-CATSWGLNEQYF (SEQ ID NO: 142), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xiv) CDR1α-DSASNY (SEQ ID NO: 129), CDR2α - IRSNVGE (SEQ ID NO: 130), CDR3α-CAATSRDDMRF (SEQ ID NO: 131), CDR1β-SEHNR (SEQ ID NO: 134), CDR2β - FQNEAQ (SEQ ID NO: 135), and CDR3β - CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xv) CDR1α-DSASNY (SEQ ID NO: 129), CDR2α - IRSNVGE (SEQ ID NO: 130), CDR3α-CAATSRDDMRF (SEQ ID NO: 131), CDR1β-LNHNV (SEQ ID NO: 140), CDR2β - YYDKDF (SEQ ID NO: 141), and CDR3β-CATSWGLNEQYF (SEQ ID NO: 142), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xvi) CDR1α-TRDTTYY (SEQ ID NO: 146), CDR2α-RNSFDEQN (SEQ ID NO: 147), CDR3α - CALPDKVIF (SEQ ID NO: 148), CDR1β-SGDLS (SEQ ID NO: 156), CDR2β-YYNGEE (SEQ ID NO: 157), and CDR3β - CASSSVAGSTGELFF (SEQ ID NO: 158), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xvii) CDR1α-SIFNT (SEQ ID NO: 151), CDR2α - LYKAGEL (SEQ ID NO: 152), CDR3α - CAGLYATNKLIF (SEQ ID NO: 153), CDR1β-SGDLS (SEQ ID NO: 156), CDR2β-YYNGEE (SEQ ID NO: 157), and CDR3β - CASSSVAGSTGELFF (SEQ ID NO: 158), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xviii) CDR1α-VSNAYN (SEQ ID NO:91), CDR2α-GSKP (SEQ ID NO: 92), CDR3α-CAAPNDYKLSF (SEQ ID NO: 93), CDR1β-MNHEY (SEQ ID NO: 118), CDR2β-SMNVEV (SEQ ID NO: 119), and CDR3β-CASSTLGGELFF (SEQ ID NO: 120), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xix) CDR1α-VSGNPY (SEQ ID NO: 108), CDR2α - YITGDNLV (SEQ ID NO: 109), CDR3α - CAVRDGGATNKLIF (SEQ ID NO: 110), CDR1β-SEHNR (SEQ ID NO: 96), CDR2β - FQNEAQ (SEQ ID NO: 97), and CDR3β - CASSSGLAFYEQYF (SEQ ID NO: 98), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xx) CDR1α-VSGNPY (SEQ ID NO: 108), CDR2α - YITGDNLV (SEQ ID NO: 109), CDR3α - CAVRDGGATNKLIF (SEQ ID NO: 110), CDR1β-SGHDN (SEQ ID NO: 102), CDR2β-FVKESK (SEQ ID NO: 103), and CDR3β - CASSQLSGRDSYEQYF (SEQ ID NO: 104), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xxi) CDR1α-NIATNDY (SEQ ID NO: 113), CDR2α - GYKTK (SEQ ID NO: 114), CDR3α - CLVGGYTGGFKTIF (SEQ ID NO: 115), CDR1β-SEHNR (SEQ ID NO: 96), CDR2β - FQNEAQ (SEQ ID NO: 97), and CDR3β - CASSSGLAFYEQYF (SEQ ID NO: 98), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xxii) CDR1α-NIATNDY (SEQ ID NO: 113), CDR2α - GYKTK (SEQ ID NO: 114), CDR3α - CLVGGYTGGFKTIF (SEQ ID NO: 115), CDR1β-SGHDN (SEQ ID NO: 102), CDR2β-FVKESK (SEQ ID NO: 103), and CDR3β - CASSQLSGRDSYEQYF (SEQ ID NO: 104), or variants thereof each having up to three amino acid substitutions, additions or deletions; or (xxiii) CDR1α-DRGSQS (SEQ ID NO: 182), CDR2α-IYSNGD (SEQ ID NO: 183), CDR3α - CASGGGADGLTF (SEQ ID NO: 25), CDR1β-SGDLS (SEQ ID NO: 28), CDR2β-YYNGEE (SEQ ID NO: 29), and CDR3β-CASGRGDTEAFF (SEQ ID NO: 30), or variants thereof each having up to three amino acid substitutions, additions or deletions.

In one embodiment, the TCR comprises: (i) an alpha chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 26 or is at least 75%, at least 80%, at least 85%, at least 90%, Variants thereof that have at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amine of SEQ ID NO: 31 The amino acid sequence or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity with it Variants thereof; (ii) α chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 4 or is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, Variants thereof that have at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 9 or Variants thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity ; (iii) α chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 15 or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, Variants thereof that have at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a β-chain variable domain comprising the amino acid sequence of SEQ ID NO: 20 or having at least 75% sequence identity therewith. %, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (iv) An alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 37 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, Variants thereof that have at least 98% or at least 99%, preferably at least 75% sequence identity; and a β-chain variable domain comprising the amino acid sequence of SEQ ID NO: 42 or having at least 75% or at least 80% sequence identity therewith. %, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (v) Alpha chain variable A domain comprising the amino acid sequence of SEQ ID NO: 37 or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or Variants thereof that have at least 99%, preferably at least 75% sequence identity; and a β-chain variable domain comprising the amino acid sequence of SEQ ID NO: 53 or having at least 75%, at least 80%, or at least 85% sequence identity with it. %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (vi) α chain variable domain, which Comprising the amino acid sequence of SEQ ID NO: 48 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, Preferably, its variant has at least 75% sequence identity; and a beta chain variable domain, which includes the amino acid sequence of SEQ ID NO: 53 or has at least 75%, at least 80%, at least 85%, or at least 90% of the amino acid sequence of SEQ ID NO: 53. %, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75%, variants thereof with sequence identity; (vii) α chain variable domain, comprising SEQ ID NO : 48 amino acid sequence or at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% % sequence identity thereof; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 42 or having at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the amino acid sequence of SEQ ID NO: 42. %, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (viii) an alpha chain variable domain, comprising the amine of SEQ ID NO: 94 The amino acid sequence or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity with it Variants thereof; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 99 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 96% of the amino acid sequence of SEQ ID NO: 99. %, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity of its variants; (ix) α-chain variable domain, which includes the amino acid sequence of SEQ ID NO: 94 or Variants thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity ; And a beta chain variable domain, which includes the amino acid sequence of SEQ ID NO: 105 or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% thereof; %, at least 98% or at least 99%, preferably at least 75% sequence identity; (x) α-chain variable domain, which includes the amino acid sequence of SEQ ID NO: 111 or has at least 75% sequence identity with it. %, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75%, sequence identity thereof; and the beta chain A variable domain comprising the amino acid sequence of SEQ ID NO: 121 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% therewith. % or at least 99%, preferably at least 75% sequence identity of its variants; (xi) α-chain variable domain, which includes the amino acid sequence of SEQ ID NO: 116 or has at least 75%, at least 80% sequence identity with it. %, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75%, sequence identity thereof; and a beta chain variable domain , which contains the amino acid sequence of SEQ ID NO: 121 or is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% %, preferably variants thereof with at least 75% sequence identity; (xii) α-chain variable domain, which includes the amino acid sequence of SEQ ID NO: 127 or has at least 75%, at least 80%, or at least 85% of the amino acid sequence of SEQ ID NO: 127. %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75%, variants thereof with sequence identity; and a beta chain variable domain comprising SEQ The amino acid sequence of ID NO: 137 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably Variants thereof with at least 75% sequence identity; (xiii) α-chain variable domain, which includes the amino acid sequence of SEQ ID NO: 127 or has at least 75%, at least 80%, at least 85%, or at least 90% similarity therewith. %, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75%, sequence identity variants thereof; and a beta chain variable domain comprising SEQ ID NO: 143 The amino acid sequence or it has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence Variants thereof; (xiv) α chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 132 or is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical thereto; %, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity variants thereof; and a beta chain variable domain comprising the amino acid of SEQ ID NO: 137 sequence or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity with it Variant; (xv) α chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 132 or is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% identical thereto. %, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity variants thereof; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 143 or having the same Variants thereof that have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; ( xvi) Alpha chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 149 or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% therewith %, at least 98% or at least 99%, preferably at least 75%, variants thereof with sequence identity; and a β-chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 or having at least 75%, Variants thereof that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xvii) α chain A variable domain comprising the amino acid sequence of SEQ ID NO: 154 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% therewith. % or at least 99%, preferably at least 75%, sequence identity variants thereof; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 159 or having at least 75%, at least 80%, Variants thereof that have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xviii) α chain variable domain , which contains the amino acid sequence of SEQ ID NO: 94 or is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% %, preferably variants thereof with at least 75% sequence identity; and a β-chain variable domain comprising the amino acid sequence of SEQ ID NO: 121 or having at least 75%, at least 80%, at least 85%, Variants thereof that have at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xix) α-chain variable domain, comprising SEQ The amino acid sequence of ID NO: 111 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably Variants thereof that have at least 75% sequence identity; and a β-chain variable domain comprising the amino acid sequence of SEQ ID NO: 99 or having at least 75%, at least 80%, at least 85%, at least 90%, Variants thereof having at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xx) α-chain variable domain, comprising SEQ ID NO: 111 The amino acid sequence or it has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence Variants thereof that are identical; and a β-chain variable domain comprising the amino acid sequence of SEQ ID NO: 105 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, Variants thereof that have at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xxi) α-chain variable domain, comprising the amino acid of SEQ ID NO: 116 sequence or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity with it Variant; and a beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 99 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, Variants thereof that have at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xxii) α-chain variable domain, which includes the amino acid sequence of SEQ ID NO: 116 or has the same amino acid sequence as SEQ ID NO: 116. Variants thereof that have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and A beta chain variable domain comprising the amino acid sequence of SEQ ID NO: 105 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, A variant thereof with at least 98% or at least 99%, preferably at least 75% sequence identity; or (xxiii) an alpha chain variable domain comprising an amino acid selected from the group consisting of SEQ ID NO: 185, 190 sequence or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity with it Variants; and beta chain variable domains, which comprise the amino acid sequence of SEQ ID NO: 31 or have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, Variants thereof that have at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity. In one embodiment, the TCR comprises: (i) an alpha chain, which comprises or is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical to the amino acid sequence of SEQ ID NO: 27, Variants thereof that have at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain comprising a member selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 203 amino acid sequences and amino acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% , preferably variants of SEQ ID NO: 32, 33 and 203 with at least 75% sequence identity; (ii) α chain, which contains the amino acid sequence of SEQ ID NO: 5 or has at least 75%, at least 80% sequence identity with it. %, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75%, variants thereof with sequence identity; and a beta chain, which includes a selected The amino acid sequence of the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 195 and having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least Variants of SEQ ID NO: 10, 11 and 195 with 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (iii) an alpha chain, comprising SEQ ID NO: 16 The amino acid sequence or it has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence Variants thereof that are identical; and a β chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 197 and has at least 75%, at least 80% , variants of SEQ ID NO: 21, 22 and 197 with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity. ; (iv) α chain, which contains the amino acid sequence of SEQ ID NO: 38 or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, Variants thereof that have at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain comprising a member selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 215 Amino acid sequence and its sequence identity is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% Variants of SEQ ID NO: 43, 44 and 215; (v) α chain, which contains the amino acid sequence of SEQ ID NO: 38 or is at least 75%, at least 80%, at least 85%, at least 90% different from the amino acid sequence of SEQ ID NO: 38. %, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75%, variants thereof with sequence identity; and a beta chain comprising a member selected from the group consisting of SEQ ID NO: 54, SEQ The amino acid sequence of the group consisting of ID NO: 55 and SEQ ID NO: 217 and its amino acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least Variants of SEQ ID NO: 54, 55 and 217 with 98% or at least 99%, preferably at least 75% sequence identity; (vi) alpha chain, which contains or has the amino acid sequence of SEQ ID NO: 49 Variants thereof that have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and β chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 217 and has at least 75%, at least 80%, at least 85%, and at least 90% similarity therewith. , variants of SEQ ID NO: 54, 55 and 217 with at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (vii) α chain, which Comprising the amino acid sequence of SEQ ID NO: 49 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, Preferably, its variant with at least 75% sequence identity; and a β chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 215 and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity to SEQ ID NO: 43, Variants of 44 and 215; (viii) α chain, which contains the amino acid sequence of SEQ ID NO: 95 or is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% identical thereto. %, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity of its variants; and a beta chain, which includes a member selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 101 Amino acid sequence and its sequence identity is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% Variants of SEQ ID NO: 100 and 101; (ix) α chain, which contains the amino acid sequence of SEQ ID NO: 95 or is at least 75%, at least 80%, at least 85%, at least 90%, Variants thereof that have at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain comprising a member selected from the group consisting of SEQ ID NO: 106, SEQ ID NO : Amino acid sequence of a group consisting of 107 and an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or more Preferably, variants of SEQ ID NO: 106 and 107 with at least 75% sequence identity; (x) α chain, which contains the amino acid sequence of SEQ ID NO: 112 or has at least 75%, at least 80%, or at least 85% sequence identity with it. %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75%, variants thereof with sequence identity; and a beta chain comprising a member selected from the group consisting of SEQ ID NO. : 122. The amino acid sequence of the group consisting of SEQ ID NO: 123 and its amino acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% Or variants of SEQ ID NO: 122 and 123 with at least 99%, preferably at least 75% sequence identity; (xi) α chain, which contains the amino acid sequence of SEQ ID NO: 117 or has at least 75%, Variants thereof that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain, which Comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 122 and SEQ ID NO: 123 and having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least Variants of SEQ ID NO: 122 and 123 with 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 128 or Variants thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity ; and a beta chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 138 and SEQ ID NO: 139 and has at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% of the amino acid sequence therewith. , variants of SEQ ID NO: 138 and 139 with at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xiii) alpha chain, comprising SEQ ID NO: 128 The amino acid sequence or it has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence Variants thereof that are identical; and a β-chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 144 and SEQ ID NO: 145 and having at least 75%, at least 80%, at least 85%, or at least Variants of SEQ ID NO: 144 and 145 having 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xiv) an alpha chain, which Comprising the amino acid sequence of SEQ ID NO: 133 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, Preferably, its variant has at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 138 and SEQ ID NO: 139 and has at least 75% and at least 80% amino acid sequence identity therewith. , variants of SEQ ID NO: 138 and 139 with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; ( xv) α chain, which contains the amino acid sequence of SEQ ID NO: 133 or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% therewith % or at least 99%, preferably at least 75% sequence identity of its variants; and a beta chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 144, SEQ ID NO: 145 and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity to SEQ ID NO: 144 and Variants of 145; (xvi) α chain, which contains the amino acid sequence of SEQ ID NO: 150 or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, Variants thereof that have at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain comprising an amine group selected from the group consisting of SEQ ID NO: 160, SEQ ID NO: 161 acid sequence and having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity therewith Variants of SEQ ID NO: 160 and 161; (xvii) α chain, which contains the amino acid sequence of SEQ ID NO: 155 or is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical thereto. %, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity variants thereof; and a beta chain comprising a member selected from the group consisting of SEQ ID NO: 160, SEQ ID NO: 161 The amino acid sequence of the group consisting of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least Variants of SEQ ID NO: 160 and 161 with 75% sequence identity; (xviii) α chain, which contains the amino acid sequence of SEQ ID NO: 95 or has at least 75%, at least 80%, at least 85%, Variants thereof that have at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain comprising a member selected from the group consisting of SEQ ID NO: 122 , the amino acid sequence of the group consisting of SEQ ID NO: 123 and having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least Variants of SEQ ID NO: 122 and 123 with 99%, preferably at least 75% sequence identity; (xix) α chain, which contains the amino acid sequence of SEQ ID NO: 112 or has at least 75%, at least 80% sequence identity with it. %, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75%, variants thereof with sequence identity; and a beta chain, which includes a selected The amino acid sequence of the group consisting of SEQ ID NO: 100 and SEQ ID NO: 101 and the amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% therewith , variants of SEQ ID NO: 100 and 101 with at least 98% or at least 99%, preferably at least 75% sequence identity; (xx) α chain, which includes or has the amino acid sequence of SEQ ID NO: 112 Variants thereof that have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and β chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 106 and SEQ ID NO: 107 and has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least Variants of SEQ ID NO: 106 and 107 with 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xxi) an alpha chain comprising the amine of SEQ ID NO: 117 The amino acid sequence or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity with it Variants thereof; and a β chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 100 and SEQ ID NO: 101 and has at least 75%, at least 80%, at least 85%, and at least 90% of the amino acid sequence therewith. , variants of SEQ ID NO: 100 and 101 with at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xxii) an alpha chain, comprising SEQ The amino acid sequence of ID NO: 117 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably Variants thereof that have at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 106, SEQ ID NO: 107 and has at least 75%, at least 80%, or at least Variants of SEQ ID NO: 106 and 107 that have 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; or (xxiii ) (a) α chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 186, 191, 198, 199, 200, 201, 202 and has at least 75%, at least 80%, and at least 85% , SEQ ID NOs: 186, 191, 198, 199, 200, 201 and at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity. Variants of 202; and a beta chain comprising the amino acid sequence of SEQ ID NO: 32 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99%, preferably at least 75% sequence identity of its variants; (b) α chain, which includes a member selected from the group consisting of SEQ ID NO: 186, 191, 198, 199, 200, 201, 202 The amino acid sequence of the group consisting of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least Variants of SEQ ID NO: 186, 191, 198, 199, 200, 201 and 202 with 75% sequence identity; and β-strands comprising or having at least 75% sequence identity with the amino acid sequence of SEQ ID NO: 33. Variants thereof that have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; or (c) α A chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 186, 191, 198, 199, 200, 201, 202 and having at least 75%, at least 80%, at least 85%, at least 90%, Variants of SEQ ID NO: 186, 191, 198, 199, 200, 201 and 202 having at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; And a beta chain, which contains the amino acid sequence of SEQ ID NO: 203 or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% therewith. Or a variant thereof with at least 99%, preferably at least 75% sequence identity. (xxiv) Alpha chain, which contains the amino acid sequence of SEQ ID NO: 194 or is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least Variants thereof with 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain comprising an amine selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 195 The amino acid sequence and its sequence identity are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75%. Variants of SEQ ID NO: 10, 11 and 195; (xxv) α chain, which contains the amino acid sequence of SEQ ID NO: 196 or has at least 75%, at least 80%, at least 85%, at least 90% of the amino acid sequence of SEQ ID NO: 196 , a variant thereof with at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain comprising a member selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, the amino acid sequence of the group consisting of SEQ ID NO: 197 and the amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98 % or at least 99%, preferably at least 75%, sequence identity of SEQ ID NO: 21, 22 and 197 variants; (xxvi) α chain, which contains the amino acid sequence of SEQ ID NO: 214 or has at least Variants thereof that have 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and β A chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 215 and having at least 75%, at least 80%, at least 85%, at least 90%, Variants of SEQ ID NO: 43, 44 and 215 having at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; (xxvii) an alpha chain, comprising The amino acid sequence of SEQ ID NO: 214 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or more Preferably, variants thereof with at least 75% sequence identity; and a β-chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 217 and having at least 75% sequence identity therewith. %, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity to SEQ ID NOs: 54, 55 and variants of 217; (xxviii) α chain, which contains the amino acid sequence of SEQ ID NO: 216 or is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% identical thereto , a variant thereof with at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity; and a beta chain comprising a sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: The amino acid sequence of the group consisting of 217 and its amino acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably Variants of SEQ ID NO: 54, 55 and 217 that have at least 75% sequence identity; or (xxix) an alpha chain, which contains the amino acid sequence of SEQ ID NO: 216 or has at least 75%, at least 80%, Variants thereof that have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, preferably at least 75% sequence identity; and a beta chain comprising a sequence selected from the group consisting of SEQ. The amino acid sequence of the group consisting of ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 215 and having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , variants of SEQ ID NO: 43, 44 and 215 with at least 97%, at least 98% or at least 99%, preferably at least 75% sequence identity.

In one embodiment, the TCR of the invention binds to a Wilms' tumor 1 protein (WT1) peptide when presented via the major histocompatibility complex (MHC), wherein the WT1 peptide comprises a protein selected from the group consisting of GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID NO: 82) and CMTWNQMNLGATLKG (SEQ ID NO: 87), and their respective variants with up to three amino acid substitutions, additions or deletions.

In one embodiment, the invention provides a T cell receptor (TCR) that binds to Wilms' tumor 1 protein (WT1) peptide when presented via the major histocompatibility complex (MHC), wherein the WT1 The peptide includes an amino acid sequence selected from the group consisting of GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID NO: 82) and CMTWNQMNLGATLKG (SEQ ID NO: 87) and each having up to three amino acid substitutions, Variations with additions or deletions.

In one embodiment, the TCR binds to MHC I and/or MHC II peptide complexes.

In one embodiment, the TCR is restricted to human leukocyte antigen (HLA) alleles. In one embodiment, the TCR is limited to HLA-A, HLA-B or HLA-C alleles. In one embodiment, the TCR is limited to HLA-A*02:01, HLA-B*38:01, HLA-C*03:03, or HLA-C*07:02.

In one embodiment, the TCR is limited to HLA-A*02:01. In one embodiment, the TCR is limited to HLA-B*38:01. In one embodiment, the TCR is limited to HLA-C*03:03. In one embodiment, the TCR is limited to HLA-C*07:02.

In one embodiment, the TCRs of the invention are limited to HLA-C alleles. In one embodiment, the TCR of the invention is limited to HLA-C alleles selected from the group consisting of: HLA-C*07:01, HLA-C*03:04, HLA-C*04:01, HLA -C*05:01, HLA-C*06:02 and HLA-C*07:02.

In one embodiment, the TCR contains one or more mutations at the alpha/beta chain interface such that when the alpha and beta chains are expressed in T cells, there is a gap between these chains and the endogenous TCR alpha and beta chains. The distribution frequency is reduced.

In one embodiment, the TCR contains one or more mutations at the alpha chain/beta chain interface such that the expression of the TCR alpha and beta chains is increased when the alpha and beta chains are expressed in T cells.

In one embodiment, one or more mutations introduce a cysteine residue into the constant region domain of each of the alpha chain and the beta chain, wherein the cysteine residue can be present in the alpha chain and the beta chain. disulfide bonds are formed between them.

In one embodiment, the one or more mutations are at amino acid positions selected from those disclosed in: Table 1 of Boulter, J.M et al. (2003) Protein Engineering 16: 707-711.

In one embodiment, the TCR contains one or more mutations to remove one or more N-glycosylation sites (see, eg, Kuball, J et al. (2009) J Exp Med 206: 463-75). Preferably, the N-glycosylation site is in the TCR constant domain. In one embodiment, the mutation is the replacement of amino acid N with amino acid Q in the N-X-S/T motif. For example, substitutions may occur at one or more of the following positions: TCR α C gene position 36, 90, or 109; and/or TCR β C gene position 85.6. Preferably, the substitution is at position 36 of the TCR α C gene.

In one embodiment, the TCR contains a murineized constant region.

In one embodiment, the TCR is a soluble TCR.

In another aspect, the invention provides an isolated polynucleotide encoding the alpha chain of a T cell receptor (TCR) according to the invention and/or the beta chain of a TCR according to the invention.

In one embodiment, the polynucleotide encodes an alpha chain linked to a beta chain.

In one embodiment, the isolated polynucleotide further encodes one or more short interfering RNA (siRNA) or other agents capable of reducing or preventing expression of one or more endogenous TCR genes.

In another aspect, the invention provides a vector comprising a polynucleotide according to the invention. In one embodiment, the vector includes a polynucleotide encoding one or more CD3 chains, CD8, a suicide gene, and/or a selectable marker.

In another aspect, the invention provides cells comprising a TCR of the invention, a polynucleotide of the invention, or a vector of the invention.

In one embodiment, the cells further comprise vectors encoding one or more CD3 chains, CD8, suicide genes and/or selectable markers.

In one embodiment, the cell line is a T cell, lymphocyte or stem cell, optionally wherein the T cell, lymphocyte or stem cell line is selected from the group consisting of: CD4 cells, CD8 cells, naive T cells, memory stem T cells, central Memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, γ/δ T cells, Natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, hematopoietic stem cells and pluripotent stem cells.

In one embodiment, the cell line is a T cell isolated from an individual.

In one embodiment, the endogenous gene encoding the TCR alpha chain and/or the endogenous gene encoding the TCR beta chain in the cell is destroyed, preferably such that the endogenous gene encoding the TCR alpha chain and/or the endogenous gene encoding the TCR beta chain Genes don't express themselves. In one embodiment, the endogenous gene encoding the TCR alpha chain and/or the endogenous gene encoding the TCR beta chain is disrupted by inserting a expression cassette comprising a polynucleotide sequence encoding the TCR of the invention. In one embodiment, one or more endogenous genes encoding MHC are disrupted, preferably where the cell line is non-alloreactive pluripotent T cells. In one embodiment, endogenous genes involved in persistence, expansion, activity, resistance to exhaustion/senescence/inhibitory signals, homing ability, or other T cell functions are disrupted, preferably among which genes involved in persistence, expansion Endogenous genes for proliferation, activity, resistance to exhaustion/senescence/inhibitory signals, homing ability or other T cell functions are selected from the group consisting of: PD1 , TIM3 , LAG3 , 2B4 , KLRG1 , TGFbR , CD160 , TIGIT , CTLA4 and CD39 . In one embodiment, endogenous genes involved in persistence, expansion, activity, resistance to exhaustion/senescence/inhibitory signals, homing ability, or other T cell functions are disrupted by integration of an expression cassette, wherein the expression cassette includes Polynucleotide sequence encoding the TCR of the invention.

In another aspect, the invention provides a method of preparing cells, the method comprising the step of introducing a vector of the invention into the cells in vitro, ex vivo or in vivo, for example by transfection or transduction.

In another aspect, the invention provides a method of preparing cells, the method comprising the step of transducing the cells in vitro, ex vivo or in vivo with one or more vectors of the invention.

In one embodiment, the cell line to be transduced with one or more vectors is selected from the group consisting of: T cells, lymphocytes or stem cells, such as hematopoietic stem cells or induced pluripotent stem cells (iPS), optionally T cells, Lymphocytes or stem cells can be selected from the following groups: CD4 cells, CD8 cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, γ/δ T cells, natural killer cells (NK) cells, natural killer T (NKT) cells, double negative T cells, naive T cells, memory stem T cells, central memory T cells, effector memory T cells, effector T cells, cytokine-induced killer (CIK) cells, hematopoietic stem cells and pluripotent stem cells.

In one embodiment, the method includes a step of T cell editing, which step includes using an artificial nuclease to destroy the endogenous gene encoding the TCR alpha chain and/or the endogenous gene encoding the TCR beta chain, preferably wherein the artificial nuclease The system is selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas systems.

In one embodiment, the method includes a step of T cell editing, which step includes using an artificial nuclease to destroy the endogenous gene encoding the TCR alpha chain and/or the endogenous gene encoding the TCR beta chain, preferably wherein the artificial nuclease The system is selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas systems.

In one embodiment, the method includes the step of targeted integration of an expression cassette into an endogenous gene encoding a TCR alpha chain and/or an endogenous gene encoding a TCR beta chain destroyed by an artificial nuclease, wherein the expression cassette includes the encoding The polynucleotide sequence of the TCR of the present invention or the polynucleotide sequence of the present invention.

In one embodiment, the method includes the step of disrupting one or more endogenous genes encoding MHC, preferably wherein the cell line produced by the method is non-alloreactive pluripotent T cells.

In one embodiment, the method includes the step of disrupting one or more endogenous MHC genes, preferably wherein the cell line produced by the method is non-alloreactive pluripotent T cells.

In one embodiment, the method includes the step of disrupting one or more endogenous genes to modify persistence, expansion, activity, resistance to exhaustion/senescence/inhibitory signals, homing ability, or other T cell function, Preferably wherein the method comprises integrating the expression cassette into an endogenous protein involved in persistence, expansion, activity, resistance to exhaustion/senescence/inhibitory signals, homing ability or other T cell functions and destroyed by an artificial nuclease The steps in the gene, wherein the expression cassette contains a polynucleotide sequence encoding the TCR of the invention, preferably wherein the endogenous gene system is selected from the group consisting of: PD1 , TIM3 , LAG3 , 2B4 , KLRG1 , TGFbR , CD160 , TIGIT , CTLA4 and CD39 .

In another aspect, the invention provides cells of the invention or cells prepared by the method of the invention for use in adoptive cell transfer, preferably adoptive T cell transfer, optionally wherein the adoptive T cell transfer is of the same species. Allogeneic adoptive T cell transfer, autologous adoptive T cell transfer, or universal non-alloreactive adoptive T cell transfer.

In another aspect, the invention provides chimeric molecules comprising the TCR of the invention, or portions thereof, coupled to non-cellular substrates, toxins and/or antibodies. In one embodiment, the non-cellular substrate is selected from the group consisting of nanoparticles, exosomes, and other non-cellular substrates.

In another aspect, the invention provides a TCR of the invention, an isolated polynucleotide of the invention, a vector of the invention, a cell of the invention, a cell prepared by a method of the invention, or a chimeric molecule of the invention, It is used in therapy.

In another aspect, the invention provides a TCR of the invention, an isolated polynucleotide of the invention, a vector of the invention, a cell of the invention, a cell prepared by a method of the invention, or a chimeric molecule of the invention, It is used to treat and/or prevent diseases associated with manifestations of WT1.

In another aspect, the invention provides genetically engineered (e.g., genetic editing) to modify persistence, expansion, activity, resistance to exhaustion/senescence/inhibitory signals, homing ability, or other T cell functions. T cells, wherein the T cells express the TCR alpha chain of the present invention and/or the TCR beta chain of the present invention.

In another aspect, the invention provides T cells genetically engineered (e.g., genetically edited) by a protocol that includes integrating an expression cassette to participate in persistence, expansion, activity, resistance to exhaustion/senescence/suppression The step in the endogenous gene that signals resistance, homing ability or other T cell functions and is destroyed by artificial nuclease, wherein the expression cassette contains a polynucleoside encoding the TCR α chain of the invention and/or the TCR β chain of the invention acid sequence.

In another aspect, the invention provides a method of treating and/or preventing a disease associated with expression of WT1, the method comprising administering to an individual in need thereof a TCR of the invention, an isolated polynucleotide of the invention, The steps of the vector of the present invention, the cells of the present invention, the cells prepared by the method of the present invention or the chimeric molecules of the present invention.

In one embodiment, the disease associated with expression of WT1 is a proliferative disorder. Preferably, the proliferative disorder is a hematological malignancy or a solid tumor. Preferably, the hematological malignancy is selected from the group consisting of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), lymphoblastic leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, Non-Hodgkin lymphoma and Hodgkin lymphoma. Preferably, the solid tumor is selected from the group consisting of: lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, bile duct cancer, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid Carcinoma, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal cancer, soft tissue sarcoma, urothelial cancer, biliary tract cancer, glioblastoma, cervical cancer, mesothelial cancer tumors and colorectal cancer.

In a preferred embodiment, the disease associated with manifestations of WT1 is acute myelogenous leukemia (AML).

In another preferred embodiment, the disease associated with the expression of WT1 is chronic myelogenous leukemia (CML).

In another aspect, the invention provides an isolated immunogenic WT1 peptide comprising an amino acid sequence selected from the group consisting of: GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID NO: 82), and CMTWNQMNLGATLKG (SEQ ID NO: 87) and its variants each having up to three amino acid substitutions, additions or deletions.

As used herein, the terms "comprising, composeds and composed of" are synonyms with "including or includes", or "containing or contains" and are inclusive or inclusive and do not exclude additional non-limiting elements or steps. The term "comprising, composeds and composed of" also includes the term "composed of".

T cell receptor During antigen processing, the antigen is degraded inside the cell and then carried to the cell surface by major histocompatibility complex (MHC) molecules. T cells can recognize this peptide:MHC complex on the surface of antigen-presenting cells. There are two different classes of MHC molecules: MHC I and MHC II, each class delivering peptides from different cellular compartments to the cell surface.

T cell receptors (TCRs) are molecules found on the surface of T cells that are responsible for recognizing antibodies that bind to MHC molecules. Natural TCR heterodimers are composed of alpha (α) and beta (β) chains in approximately 95% of T cells, while approximately 5% of T cells have gamma (γ) and delta (δ) chains. of TCR.

Engagement of the TCR with antigen and MHC activates T lymphocytes, on which the TCR manifests through a series of biochemical events mediated by associated enzymes, coreceptors, and specialized accessory molecules.

Each chain of natural TCR is a member of the immunoglobulin superfamily and has an N-terminal immunoglobulin (Ig)-variable (V) domain, an Ig-constant (C) domain, and spans the membrane/cell membrane. region and a short C-terminal cytoplasmic tail.

The variable domains of TCR alpha and beta chains have three hypervariable or complementarity determining regions (CDRs). The TCR alpha or beta chain, for example, includes CDR1, CDR2 and CDR3 in amine to carboxyl terminus order. Generally speaking, CDR3 is the main CDR responsible for recognition of the antigen being processed, but CDR1 of the alpha chain has also been shown to interact with the N-terminal portion of the antigenic peptide, while CDR1 of the beta chain interacts with the C-terminal portion of the peptide. CDR2 is believed to recognize MHC molecules.

The constant domain of a TCR can consist of short linker sequences in which cysteine residues form disulfide bonds, creating a link between the two chains.

The alpha chain of the TCR of the present invention may have a constant domain encoded by the TRAC gene. An exemplary amino acid sequence of the alpha chain constant domain encoded by the TRAC gene is shown below: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 76)

The TCR of the present invention may comprise an alpha chain, which contains the amino acid sequence of SEQ ID NO: 76 or is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 97% identical thereto. %, at least 98%, at least 99% sequence identity, preferably at least 75% sequence identity with its variants.

The β chain of the TCR of the present invention may have a constant domain encoded by the TRBC1 or TRBC2 gene. An exemplary amino acid sequence of the beta chain constant domain encoded by the TRBC1 gene is shown below: DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 77)

An exemplary amino acid sequence of the beta chain constant domain encoded by the TRBC2 gene is shown below: DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 78)

The TCR of the present invention may comprise a β chain, which contains the amino acid sequence of SEQ ID NO: 77, SEQ ID NO: 78 or has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, Variants of SEQ ID NOs: 77 and 78 having at least 96%, at least 97%, at least 98%, at least 99% sequence identity, preferably at least 75% sequence identity thereto.

TCRs of the invention may have one or more additional cysteine residues in each of the alpha and beta chains, such that the TCR may contain two or more disulfide bonds in the constant domain.

Mutations in the TCR constant domains disclosed herein may be illustrated based on a numbering convention in which the first amino acid of each of SEQ ID NOs: 76-78 is assigned to position 2.

This structure allows the TCR to associate with other molecules (such as CD3) that have three different chains in mammals (γ, δ, and ε) and ζ-chains. These accessory molecules have negatively charged transmembrane regions and are critical for propagating signals from the TCR into the cell. CD3- and ζ-chains together with TCR form what is called a T cell receptor complex.

The signal from the T cell complex is enhanced by simultaneous binding of MHC molecules by specific coreceptors. For helper T cells, this coreceptor system is CD4 (specific for MHC class II); for cytotoxic T cells, this coreceptor system is CD8 (specific for MHC class I). Coreceptors allow prolonged engagement between antigen-presenting cells and T cells and recruit intracellular basic molecules (eg, LCK) involved in signaling of activated T lymphocytes.

Thus, the term "T cell receptor" (TCR) as used herein refers to a molecule capable of recognizing a peptide when presented by an MHC molecule. The molecule may be a heterodimer of two chains α and β (or γ and δ as appropriate), or it may be a single chain TCR construct. The TCR of the invention can be a soluble TCR, for example, one or more constant domains are omitted or altered. The TCR of the invention may comprise a constant domain.

The invention also provides alpha or beta chains from the T cell receptor.

The TCR of the invention may be a hybrid TCR comprising sequences derived from more than one species. For example, it has been found that murine TCRs are expressed more efficiently in human T cells than in human TCRs. Thus, the TCR may comprise murine sequences within the human variable and constant regions.

The disadvantage of this approach is that the murine constant sequence can trigger an immune response, leading to rejection of the transferred T cells. However, control protocols used to prepare patients for adoptive T cell therapy can be parameterized to be sufficiently immunosuppressive to allow engraftment of T cells expressing murine sequences.

In one embodiment, the TCR contains one or more mutations to remove one or more N-glycosylation sites. Preferably, the N-glycosylation site is in the TCR constant domain. Deletion of N-glycosylation sites in TCR constant domains is described in Kuball, J et al. (2009) J Exp Med 206: 463-75. In one embodiment, one or more mutations replace amino acid N in the N-X-S/T motif with amino acid Q. For example, substitutions may be at one or more of the following positions: TCR α C gene position 36, 90, or 109; and/or TCR β C gene position 85.6. Preferably, the substitution is at position 36 of the TCR α C gene.

Complementarity Determining Regions (CDR) Establish that the portion of the TCR that contacts most antigenic peptides that bind to the major histocompatibility complex (MHC) is complementarity determining region 3 (CDR3), which is unique to each T cell lineage. . CDR3 regions are generated when somatic rearrangements occur in the thymus and involve non-contiguous genes belonging to the variable (V), diversity (D, for beta and delta chains), and junction (J) genes. In addition, insertion/deletion of random nucleotides at the rearrangement locus of each TCR chain gene greatly increases the diversity of highly variable CDR3 sequences. Therefore, the frequency of a particular CDR3 sequence in a biological sample is indicative of the abundance of a particular T cell population. The great diversity of TCR profiles in healthy humans provides broad-spectrum protection against a variety of foreign antigens presented by MHC molecules on the surface of antigen-presenting cells. In this regard, it should be noted that theoretically up to 10 ¹⁵ different TCRs can be generated in the thymus.

T cell receptor diversity is concentrated in CDR3 and this region is mainly responsible for antigen recognition.

The sequence of the CDR3 region of the TCR of the present invention can be selected from those described in Table 1 below. The TCR may include a CDR that includes or consists of the CDR3α and CDR3β pair described below.

A CDR may, for example, comprise one, two or three substitutions, additions or deletions from a given sequence, provided that the TCR retains the ability to bind the WT1 peptide when presented via an MHC molecule.

The term "protein" as used herein includes single-chain polypeptide molecules as well as multiple polypeptide complexes in which the individual constituent polypeptides are linked by covalent or non-covalent means. The term "polypeptide" as used herein refers to a polymer in which amino acids are monomeric and joined together via peptide or disulfide bonds.

Variants, derivatives, analogs, homologues and fragments In addition to the specific proteins and polynucleotides mentioned herein, the invention also encompasses the use of variants, derivatives, analogs, homologues and fragments thereof.

In the context of the present invention, a variant of any given sequence is a specific sequence of its residues (amino acid or nucleic acid residues) such that the polypeptide or polynucleotide substantially retains at least one of its endogenous functions modified sequence. Variant sequences can be obtained by addition, deletion, substitution, modification, substitution and/or change of at least one residue present in the native protein.

Variant amino acid sequences of the invention, referred to as having up to three amino acid substitutions, additions or deletions, may have, for example, one, two or three amino acid substitutions, additions or deletions.

As used herein, the term "derivative" with respect to a protein or polypeptide of the invention includes any substitution, change, modification, substitution, deletion and/or addition of one (or more) amino acid residues from or to the sequence. , provided that the resulting protein or polypeptide substantially retains at least one of its endogenous functions.

The term "analog" as used herein with respect to a polypeptide or polynucleotide includes any mimetic, that is, a chemical compound that has at least one of the endogenous functions of the polypeptide or polynucleotide that it mimics.

Proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and produce functionally equivalent proteins. Conservative amino acid substitutions can be made based on similarities in polarity, charge, solubility, hydrophobic, hydrophilic and/or amphipathic properties of the residues involved, as well as retention of endogenous functionality. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and have non-polar head groups with similar hydrophilicity values. The amino acids include asparagine, glutamine, serine, threonine and tyrosine.

Substitution may involve substituting an amino acid for a similar amino acid (conservative substitution). Similar amino acids have side chain moieties that contain related properties grouped together, for example as shown below: (i) Basic side chain: lysine (K), arginine (R), histidine (H); (ii) Acidic side chains: aspartic acid (D) and glutamic acid (E); (iii) Without polar side chains: asparagine (N), glutamine (Q), serine (S), threonine (T) and tyrosine (Y); or (iv) Non-polar side chains: glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), Phenylalanine (F), methionine (M), tryptophan (W) and cysteine (C).

Any amino acid changes should maintain the ability of the TCR to bind WT1 peptide presented by MHC molecules.

Variant sequences may contain amino acid substitutions, additions, deletions and/or insertions. Changes may be concentrated in one or more regions of the alpha or beta chain (eg, constant regions), linker or framework regions, or they may be diffuse throughout the TCR molecule.

Conservative substitutions, additions or deletions may be made, for example, according to the table below. Amino acids in the same box in the second column and preferably in the same column in the third column may be substituted for each other: aliphatic non-polar GAP ILV Polarity - Uncharged CSTM NQ Polarity - Charged DE KR aromatic HFW

The present invention also encompasses homologous substitutions (both substitution and substitution are used herein to mean the exchange of an existing amino acid residue with a replacement residue), e.g., like-for-like substitutions, e.g. Base replaces base, acid replaces acid, polar replaces polar, etc. Non-homologous substitutions may also occur, for example, from one type of residue to another, or alternatively involve the inclusion of non-natural amino acids, such as ornithine.

The term "variant" as used herein may mean an entity that has some homology to a wild-type amino acid sequence or a wild-type nucleotide sequence. The term "homology" is synonymous with "identity".

Variant sequences may include amino acid sequences that are at least 50%, 55%, 65%, 75%, 85% or 90% identical to the target sequence, preferably at least 95%, at least 97% or at least 99% identical. Typically, a variant will contain an active site, etc. that is identical to the target amino acid sequence. Although homology may also be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), in the context of the present invention, homology is preferably expressed in terms of sequence identity.

Variant sequences may include nuclei that are at least 40%, 45%, 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, at least 97% or at least 99% identical to the target sequence. nucleotide sequence. Although homology may also be considered in terms of similarity, in the context of the present invention homology is preferably expressed in terms of sequence identity.

Preferably, a reference to a sequence having a certain percent identity to any of the SEQ ID NOs detailed herein refers to a sequence having said percent identity over the entire length of the SEQ ID NO mentioned. .

Identity comparisons can be performed by eye, or more commonly with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology or identity between two or more sequences.

Percent homology can be calculated on contiguous sequences, that is, aligning one sequence to another and directly comparing each amino acid in one sequence to the corresponding amino acid in the other sequence, one residue at a time. . This is called a "gap-free" alignment. Typically, these gap-free alignments are only performed on a relatively short number of residues.

Although this is an extremely simple and sequential method, it cannot take into account the following considerations: for example, in an otherwise identical sequence pair, an insertion or deletion of one nucleotide sequence may cause the following codons to not align, thus This potentially results in a significant reduction in percent homology when performing an overall alignment. Therefore, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly compromising the overall homology score. This is accomplished by inserting "gaps" in the sequence alignment in an attempt to maximize local homology.

However, these more sophisticated methods assign a "gap penalty" to each gap that occurs in the alignment so that for the same number of identical amino acids, there are as few gaps as possible (reflected in the higher number of gaps in the two compared sequences). Relevance) sequence alignments will achieve higher scores than those with many gaps. Often an "affine gap cost" is used, which pays a relatively high cost for the existence of a gap and a small penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty will of course produce the best alignment with fewer gaps. Most matching programs allow for improved gap penalties. However, when using the software for subsequent comparisons, it is better to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for an amino acid sequence is -12 for the gapped system and -4 for each extended system.

Therefore, calculation of maximum % homology first requires generating an optimal alignment, taking into account gap penalties. A suitable computer program for performing this alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software that can perform sequence comparisons include (but are not limited to) the BLAST package (see Ausubel et al. (1999) supra - Chapter 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410 ) and GENEWORKS compare covalent packages. Both BLAST and FASTA can be used for offline and online searches (see Ausubel et al. (1999) supra, pp. 7-58 to 7-60). However, for some applications it is better to use the GCG Bestfit program. Another tool called BLAST 2 Sequences can also be used to compare protein and nucleotide sequences (see FEMS Microbiol. Lett . (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8).

Although the final percent homology can be measured in terms of identity, the alignment process itself is usually not based on all-or-nothing pairwise comparisons. Instead, a scaled similarity scoring matrix is typically used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. A commonly used example of this matrix is the BLOSUM62 matrix - the default matrix of the BLAST suite of programs. GCG Wisconsin programs typically use public defaults or custom symbol comparison tables (if supplied) (see the user manual for further details). For some applications, it is better to use the public defaults of the GCG package or to use the default matrix in the case of other software, such as BLOSUM62.

Once the software generates the optimal alignment, it can calculate percent homology and optimal sequence identity. Software typically generates optimal pairs as part of sequence comparisons and generates numerical results.

"Fragment" is also a variant and the term generally refers to a selected region of a polypeptide or polynucleotide that is of interest functionally or, for example, in an analysis. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

Such variants can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where an insertion is desired, synthetic DNA encoding the insert is available along with 5' and 3' flanking regions corresponding to the native sequence on either side of the insertion site. The flanking regions will contain convenient restriction sites that correspond to positions in the native sequence so that the sequence can be cleaved with appropriate enzymes and synthetic DNA ligated to the site of cleavage. The DNA is then expressed in accordance with the invention to prepare the encoded protein. These methods not only illustrate various standard techniques known in the industry for manipulating DNA sequences, but other known techniques may also be used.

major histocompatibility complex (MHC) molecules Typically, a TCR binds to a peptide as part of a peptide:MHC complex.

MHC molecules can be MHC class I or II molecules. The complex can be on the surface of an antigen-presenting cell (eg, a dendritic cell or a B cell) or any other cell (including cancer cells), or it can be immobilized by, for example, coating on beads or a plate.

The human leukocyte antigen system (HLA) is a gene complex encoding the major histocompatibility complex (MHC) in humans and includes HLA class I antigens (A, B, and C) and HLA class II antigens (DP, DQ, and DR) The name of the body. HLA alleles A, B and C present peptides derived primarily from intracellular proteins, such as proteins expressed intracellularly. This is particularly relevant since WT1 is an intracellular protein.

During T cell development in vivo, T cells undergo positive selection steps to ensure recognition of self-MHC, followed by negative steps to remove T cells that bind too strongly to MHC presenting self-antigens. Therefore, certain T cells and the TCRs they express will only recognize peptides presented by certain types of MHC molecules, namely those encoded by specific HLA alleles. This is called HLA restriction.

One HLA allele of interest is HLA-A*0201, which is expressed in the vast majority (>50%) of the Caucasian population. Therefore, immunotherapy utilizing TCRs that bind MHC-presented WT1 peptides encoded by HLA-A*0201 (i.e., HLA-A*0201 restricted) would be suitable for treating a large proportion of the Caucasian population, so these TCRs are advantageous .

Other HLA alleles of interest are HLA-B*38:01, HLA-C*03:03 and HLA-C*07:02.

Other alleles of interest are HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA and HLA-DRB1. These are the six major MHC class II genes in humans.

In one embodiment, the TCR of the present invention is HLA-A*0201-, HLA-A*0101-, HLA-A*2402-, HLA-A*0301-, HLA-B*3501- or HLA-B* 0702 restricted.

The TCR of the present invention may be HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CILSTRVWAGSYQLTF (SEQ ID NO: 14) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CATGQATQETQYF (SEQ ID NO: 19 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CASGGGADGLTF (SEQ ID NO: 25) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASGRGDTEAFF (SEQ ID NO: 30 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSSGLAFYEQYF (SEQ ID NO: 98 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CASSQLSGRDSYEQYF (SEQ ID NO: 104 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSTLGGELFF (SEQ ID NO: 120 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSTLGGELFF (SEQ ID NO: 120 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CATSWGLNEQYF (SEQ ID NO: 142 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CAATSRDDMRF (SEQ ID NO: 131) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises a CDR3α containing the amino acid sequence of CAATSRDDMRF (SEQ ID NO: 131) or a variant thereof with up to three amino acid substitutions, additions or deletions, and a CDR3α containing CATSWGLNEQYF (SEQ ID NO: 142 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CALPDKVIF (SEQ ID NO: 148) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSVSAGSTGELFF (SEQ ID NO: 158 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CAGLYATNKLIF (SEQ ID NO: 153) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSVSAGSTGELFF (SEQ ID NO: 158 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSTLGGELFF (SEQ ID NO: 120 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSSGLAFYEQYF (SEQ ID NO: 98 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises a CDR3α containing the amino acid sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof with up to three amino acid substitutions, additions or deletions, and a CDR3α containing CASSQLSGRDSYEQYF (SEQ ID NO: 104 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSSGLAFYEQYF (SEQ ID NO: 98 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CASSQLSGRDSYEQYF (SEQ ID NO: 104 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

In one embodiment, the HLA-A*02:01 restricted TCR of the invention binds to a variant comprising the amino acid sequence LLAAILDFLLLQDPA (SEQ ID NO: 82) or having up to three amino acid substitutions, additions or deletions. body WT1 peptide.

In one aspect, the invention provides a TCR that binds a Wilms' tumor 1 protein (WT1) peptide when presented via the major histocompatibility complex (MHC), wherein the TCR comprises CILSTRVWAGSYQLTF (SEQ ID NO: 14 ), or a CDR3α of a variant thereof having up to three amino acid substitutions, additions or deletions, and an amino acid sequence comprising CATGQATQETQYF (SEQ ID NO: 19) or having up to three amino acid substitutions , CDR3β of a variant thereof with addition or deletion, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or has up to three amino acid substitutions, Variations with additions or deletions.

In one aspect, the invention provides a TCR that binds a Wilms' tumor 1 protein (WT1) peptide when presented via the major histocompatibility complex (MHC), wherein the TCR comprises CASGGGADGLTF (SEQ ID NO: 25 ), or a CDR3α of a variant thereof having up to three amino acid substitutions, additions or deletions, and an amino acid sequence comprising CASGRGDTEAFF (SEQ ID NO: 30) or having up to three amino acid substitutions , CDR3β of a variant thereof with addition or deletion, wherein the TCR is HLA-A*0201 restricted, and wherein the WT1 peptide comprises the amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or has up to three amino acid substitutions, Variations with additions or deletions.

Another widely expressed HLA allele of interest is HLA-B*38:01. The TCR of the present invention may be HLA-B*38:01 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CAMRTGGGADGLTF (SEQ ID NO: 3) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSEAGLSYEQYF (SEQ ID NO: 8 ) or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-B*38:01 restricted.

In one aspect, the invention provides a TCR that binds a Wilms' tumor 1 protein (WT1) peptide when presented via the major histocompatibility complex (MHC), wherein the TCR comprises CAMRTGGGADGLTF (SEQ ID NO: 3 ), or a CDR3α of a variant thereof having up to three amino acid substitutions, additions or deletions, and an amino acid sequence comprising CASSEAGLSYEQYF (SEQ ID NO: 8) or having up to three amino acid substitutions , CDR3β of a variant thereof with addition or deletion, wherein the TCR is HLA-B*38:01 restricted, and wherein the WT1 peptide contains the amino acid sequence of GAQYRIHTHGVFRGI (SEQ ID NO: 181) or has up to three amino acids Variations thereof with substitutions, additions or deletions.

Another widely expressed HLA allele of interest is HLA-C*07:02. The TCR of the present invention may be HLA-C*07:02 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CAMRTGGGADGLTF (SEQ ID NO: 3) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASSEAGLSYEQYF (SEQ ID NO: 8 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-C*07:02 restricted.

In one aspect, the invention provides a TCR that binds a Wilms' tumor 1 protein (WT1) peptide when presented via the major histocompatibility complex (MHC), wherein the TCR comprises CAMRTGGGADGLTF (SEQ ID NO: 3 ), or a CDR3α of a variant thereof having up to three amino acid substitutions, additions or deletions, and an amino acid sequence comprising CASSEAGLSYEQYF (SEQ ID NO: 8) or having up to three amino acid substitutions , CDR3β of a variant thereof with addition or deletion, wherein the TCR is HLA-C*07:02 restricted, and wherein the WT1 peptide comprises the amino acid sequence of GAQYRIHTHGVFRGI (SEQ ID NO: 181) or has up to three amino acids Variations thereof with substitutions, additions or deletions.

Another widely expressed HLA allele of interest is HLA-C*03:03. The TCR of the present invention may be HLA-C*03:03 restricted.

wherein the TCR of the present invention comprises CDR3α containing the amino acid sequence of CASGGGADGLTF (SEQ ID NO: 25) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CDR3α containing CASGRGDTEAFF (SEQ ID NO: 30 ), or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-C*03:03 restricted.

In one aspect, the invention provides a TCR that binds a Wilms' tumor 1 protein (WT1) peptide when presented via the major histocompatibility complex (MHC), wherein the TCR comprises CASGGGADGLTF (SEQ ID NO: 25 ), or a CDR3α of a variant thereof having up to three amino acid substitutions, additions or deletions, and an amino acid sequence comprising CASGRGDTEAFF (SEQ ID NO: 30) or having up to three amino acid substitutions , CDR3β of a variant thereof with addition or deletion, wherein the TCR is HLA-C*03:03 restricted, and wherein the WT1 peptide comprises the amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or has up to three amino acids Variations thereof with substitutions, additions or deletions.

In one embodiment wherein the TCR of the invention binds to a WT1 peptide comprising the amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-A*02:01 restricted.

In one embodiment wherein the TCR of the invention binds to a WT1 peptide comprising the amino acid sequence of GAQYRIHTHGVFRGI (SEQ ID NO: 181) or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-B*38:01 restricted.

In one embodiment wherein the TCR of the invention binds to a WT1 peptide comprising the amino acid sequence of GAQYRIHTHGVFRGI (SEQ ID NO: 181) or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-C*07:02 restricted.

In one embodiment wherein the TCR of the invention binds to a WT1 peptide comprising the amino acid sequence of LLAAILDFLLLQDPA (SEQ ID NO: 82) or a variant thereof with up to three amino acid substitutions, additions or deletions, the TCR is HLA-C*03:03 restricted.

Wilms' tumor 1 (WT1) protein Wilms' tumor 1 (WT1) is an intracellular protein encoding a zinc-finger transcription factor that plays an important role in cell growth and differentiation (Yang, L. et al. Leukemia 21, 868 -876 (2007)). It is widely expressed in a variety of blood and solid tumors, while showing restricted expression in other tissues (gonads, uterus, kidney, mesothelium, progenitor cells in different tissues). Recent evidence suggests that WT1 plays a role in leukemogenesis and tumorigenesis.

WT1 has several isoforms, some of which arise from alternative splicing of the mRNA transcript encoding WT1. The complete amino acid sequence of the WT1 isoform was previously published (Gessler, M. et al. Nature ; 343(6260):774-778; (1990)). This particular isoform consists of 575 amino acids and includes the first 126 amino acids before the N-terminus, which is lacking in the WT1 exon 5+ and KTS+ isoforms.

An example of a WT1 protein has the amino acid sequence set forth in UniProt entry J3KNN9. Another exemplary WT1 protein has the amino acid sequence set forth below: SRQRPHPGALRNPTACPLPHFPPSLPPTHSPTHPPRAGTAAQAPGPRRLLAAILDFLLLQDPASTCVPEPASQHTLRSGPGCLQQPEQQGVRDPGGIWAKLGAAEASAERLQGRRSRGASGSEPQQMGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHF SGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSA SETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL (SEQ ID NO: 79)

WT1 peptide The term peptide as used herein refers to a plurality of amino acid residues linked by peptide bonds. As defined herein, the length of the peptide can range from less than about 30, less than about 25, less than about 20, less than 19, less than 18, less than 17, less than 16, less than 15, less than 14, less than 13. Composed of less than 12, less than 11, less than 10, less than 9, less than 8, less than 7, less than 6 or less than 5 amino acid residues. Preferably, the peptide is about 5 to 20 amino acid residues in length, more preferably, the peptide is about 8 to 15 amino acid residues in length.

The TCR of the invention binds to the WT1 peptide when presented via MHC. The term WT1 peptide as used herein is understood to mean a peptide comprising an amino acid sequence derived from the WT1 protein.

For example, the WT1 peptide may comprise at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16 of the amino acid sequences of the WT1 protein. , at least 17, at least 18, at least 19, at least 20, or at least 25 contiguous amino acid residues.

The WT1 peptide may comprise an amino acid sequence selected from the group consisting of GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID NO: 82) and CMTWNQMNLGATLKG (SEQ ID NO: 87) or each having up to three amino acids Variations of substitutions, additions or deletions, or combinations thereof.

In some embodiments, for a WT1 peptide that binds to an MHC molecule encoded by the HLA-A*0201 allele, it may be preferable that the amino acid at position 2 of the peptide (i.e., the second amine from the N-terminus The amino acid) is leucine or methionine, but isoleucine, valine, alanine and threonine are also preferred. It is also preferred that the amino acid at position 9 or 10 is valine, leucine or isoleucine, but alanine, methionine and threonine are also preferred. Preferred MHC binding motifs for other HLA alleles are revealed in Celis et al. (Molecular Immunology, Vol. 31, Dec. 8, 1994, pp. 1423-1430).

The present invention encompasses various uses of the WT1 peptides described herein. For example, a WT1 peptide described herein can be administered to an individual, such as a human individual. Administration of the WT1 peptide of the present invention can trigger an immune response against cells that express or overexpress the WT1 protein, that is, the WT1 peptide is an immunogenic WT1 peptide.

Accordingly, in another aspect, the invention provides an isolated immunogenic WT1 peptide comprising a peptide selected from the group consisting of GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID NO: 82), and CMTWNQMNLGATLKG (SEQ ID NO: 87 ) and their variants each having up to three amino acid substitutions, additions or deletions.

The WT1 peptides described herein, for example, comprise an amino acid sequence selected from the group consisting of GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID NO: 82), and CMTWNQMNLGATLKG (SEQ ID NO: 87), and each has up to three amino acid sequences. Variants of WT1 peptides with amino acid substitutions, additions or deletions can be used to screen and/or identify novel TCR sequences that bind to WT1 cells. For example, T2 cells can be pulsed with the WT1 peptides mentioned in the present invention and cultured with a population of T cells isolated from the donor. In this approach, the expression of interleukins, such as CD107a and IFNγ, may indicate recognition of WT1 peptide by T cells.

Accordingly, in one aspect, the present invention provides a T cell receptor (TCR) that binds to the Wilms' tumor 1 protein (WT1) peptide when presented via the major histocompatibility complex (MHC), wherein The WT1 peptide includes an amino acid sequence selected from the group consisting of GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID NO: 82) and CMTWNQMNLGATLKG (SEQ ID NO: 87) and each having up to three amino acid substitutions , additions or deletions.

TCR sequence The amino acid sequence of the TCR that binds to the WT1 peptide described herein has been determined. Specifically, the amino acid sequences of the TCR CDRs that are important for WT1 peptide recognition and binding have been determined.

In one aspect, the invention provides a TCR comprising CDR3α containing the amino acid sequence of CILSTRVWAGSYQLTF (SEQ ID NO: 14) or a variant thereof with up to three amino acid substitutions, additions or deletions, and comprising CATGQATQETQYF CDR3β of the amino acid sequence of (SEQ ID NO: 19) or variants thereof with up to three amino acid substitutions, additions or deletions, which when presented by MHC binds to a compound containing LLAAILDFLLLQDPA (SEQ ID NO: 82) The amino acid sequence of the WT1 peptide or a variant thereof with up to three amino acid substitutions, additions or deletions.

In one aspect, the invention provides a TCR comprising a CDR3α containing the amino acid sequence of CASGGGADGLTF (SEQ ID NO: 25) or a variant thereof with up to three amino acid substitutions, additions or deletions, and comprising CASGRGDTEAFF CDR3β of the amino acid sequence of (SEQ ID NO: 30) or a variant thereof with up to three amino acid substitutions, additions or deletions, which when presented by MHC binds to a compound containing LLAAILDFLLLQDPA (SEQ ID NO: 82) The amino acid sequence of the WT1 peptide or a variant thereof with up to three amino acid substitutions, additions or deletions.

In one aspect, the invention provides a TCR comprising a CDR3α containing the amino acid sequence of CAMRTGGGADGLTF (SEQ ID NO: 3) or a variant thereof with up to three amino acid substitutions, additions or deletions, and comprising CASSEAGLSYEQYF CDR3β of the amino acid sequence of (SEQ ID NO: 8) or a variant thereof with up to three amino acid substitutions, additions or deletions, which when presented by MHC binds to a compound containing GAQYRIHTHGVFRGI (SEQ ID NO: 181) The amino acid sequence of the WT1 peptide or a variant thereof with up to three amino acid substitutions, additions or deletions.

In one aspect, the invention provides a TCR comprising CDR3α containing the amino acid sequence of CAVIGGTDSWGKLQF (SEQ ID NO: 36) or a variant thereof with up to three amino acid substitutions, additions or deletions, and comprising CASSQEEGAVYGYTF CDR3β of the amino acid sequence of (SEQ ID NO: 41) or variants thereof with up to three amino acid substitutions, additions or deletions, which when presented by MHC binds to CMTWNQMNLGATLKG (SEQ ID NO: 87) The amino acid sequence of the WT1 peptide or a variant thereof with up to three amino acid substitutions, additions or deletions.

In one aspect, the invention provides a TCR comprising a CDR3α containing the amino acid sequence of CAVIGGTDSWGKLQF (SEQ ID NO: 36) or a variant thereof with up to three amino acid substitutions, additions or deletions, and CATSREGLAADTQYF CDR3β of the amino acid sequence of (SEQ ID NO: 52) or variants thereof with up to three amino acid substitutions, additions or deletions, which when presented by MHC binds to CMTWNQMNLGATLKG (SEQ ID NO: 87) The amino acid sequence of the WT1 peptide or a variant thereof with up to three amino acid substitutions, additions or deletions.

In one aspect, the invention provides a TCR comprising a CDR3α containing the amino acid sequence of CVVPRGLSTDSWGKLQF (SEQ ID NO: 47) or a variant thereof with up to three amino acid substitutions, additions or deletions, and comprising CATSREGLAADTQYF CDR3β of the amino acid sequence of (SEQ ID NO: 52) or variants thereof with up to three amino acid substitutions, additions or deletions, which when presented by MHC binds to CMTWNQMNLGATLKG (SEQ ID NO: 87) The amino acid sequence of the WT1 peptide or a variant thereof with up to three amino acid substitutions, additions or deletions.

In one aspect, the invention provides a TCR comprising CDR3α containing the amino acid sequence of CVVPRGLSTDSWGKLQF (SEQ ID NO: 47) or a variant thereof with up to three amino acid substitutions, additions or deletions, and comprising CASSQEEGAVYGYTF CDR3β of the amino acid sequence of (SEQ ID NO: 41) or variants thereof with up to three amino acid substitutions, additions or deletions, which when presented by MHC binds to CMTWNQMNLGATLKG (SEQ ID NO: 87) The amino acid sequence of the WT1 peptide or a variant thereof with up to three amino acid substitutions, additions or deletions.

Exemplary TCR amino acid sequences of the invention are provided in Table 1. Table 1 Donor: HD12 chain district amino acid sequence SEQ ID NO Alpha (α) CDR1α TSDQSYG SEQ ID NO: 1 CDR2α QGSYDEQN SEQ ID NO: 2 CDR3α CAMRTGGGADGLTF SEQ ID NO: 3 variable MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMRTGGGADGLTFGKGTHLIIQPY SEQ ID NO: 4 Full - with TRAC constant domain MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMRTGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFAC ANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 5 Full - with TRAC constant domain MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSDQSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMRTGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFAC ANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 194 Beta (β) CDR1β SNHLY SEQ ID NO: 6 CDR2β FYNNEI SEQ ID NO: 7 CDR3β CASSEAGLSYEQYF SEQ ID NO: 8 variable MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEAGLSYEQYFPGGTRLTVTE SEQ ID NO: 9 Full - has TRBC1 constant domain MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEAGLSYEQYFPGGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 10 Full - has TRBC2 constant domain MDTWLVCWWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEAGLSYEQYFPGGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 11 Full - has TRBC2 constant domain MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEAGLSYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 195 Donor: HD13 chain district amino acid sequence SEQ ID NO Alpha (α) CDR1α TISGTDY SEQ ID NO: 12 CDR2α GLTSN SEQ ID NO: 13 CDR3α CILSTRVWAGSYQLTF SEQ ID NO: 14 variable MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILSTRVWAGSYQLTFGKGTKLSVIPN SEQ ID NO: 15 Full - with TRAC constant domain MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILSTRVWAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNN SIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 16 Full - with TRAC constant domain MKLVTSITVLLSLGIMGDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILSTRVWAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNN SIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 196 Beta (β) CDR1β KGHDR SEQ ID NO: 17 CDR2β SFdVK SEQ ID NO: 18 CDR3β CATGQATQETQYF SEQ ID NO: 19 variable MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATGQATQETQYFPGGTRLLVLE SEQ ID NO: 20 Full - has TRBC1 constant domain MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATGQATQETQYFPGGTRLLVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCL SSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 21 Full - has TRBC2 constant domain MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATGQATQETQYFPGGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCL SSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 22 Full - has TRBC2 constant domain MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPGLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATGQATQETQYFPGGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCL SSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 197 Donor: HD14 chain district amino acid sequence SEQ ID NO Alpha (α) ( with TRAV12-3*01) CDR1α nsAFQ SEQ ID NO: 23 CDR2α TYSSGN SEQ ID NO: 24 CDR3α CASGGGADGLTF SEQ ID NO: 25 variable MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCASGGGADGLTFGKGTHLIIQPY SEQ ID NO: 26 Full - with TRAC constant domain MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCASGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDF ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 27 Alpha (α) ( with TRAV12-2*01) CDR1α DRGSQS SEQ ID NO: 182 CDR2α IYSNGD SEQ ID NO: 183 CDR3α CASGGGADGLTF SEQ ID NO: 25 variable MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGTHLIIQPY SEQ ID NO: 185 Full - with TRAC constant domain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDF ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 186 Alpha (α) ( with TRAV12-2*02) CDR1α DRGSQS SEQ ID NO: 182 CDR2α IYSNGD SEQ ID NO: 183 CDR3α CASGGGADGLTF SEQ ID NO: 25 variable MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGTHLIIQPY SEQ ID NO: 190 Full - with TRAC constant domain MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKS DFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 191 Alpha (α) (TRAV12-3*01 WT) Full - with TRAC constant domain MMKSLRVLLVILWLQLSWVWSQQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCASGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 198 Alpha (α) (TRAV12-2*01 WT) Full - with TRAC constant domain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDF ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 199 Alpha (α) (TRAV12-2*01 mut) Full - with TRAC constant domain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKS DFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 200 Alpha (α) (TRAV12-2*02 WT) Full - with TRAC constant domain MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKS DFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 201 Alpha (α) (TRAV12-2*02 mut) Full - with TRAC constant domain MMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCASGGGADGLTFGKGTHLIIQPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSN KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 202 Beta (β) CDR1β SGDLS SEQ ID NO: 28 CDR2β YYNGEE SEQ ID NO: 29 CDR3β CASGRGDTEAFF SEQ ID NO: 30 variable MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASGRGDTEAFFGQGTRLTVVE SEQ ID NO: 31 Full - has TRBC1 constant domain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASGRGDTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 32 Full - has TRBC2 constant domain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASGRGDTEAFFGQGTRLTVVEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCL SSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 33 Beta (β) (TRAV12-3*01 WT) Full - has TRBC2 constant domain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASGRGDTEAFFGQGTRLTVVEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCL SSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 203 Donor: HD15 chain district amino acid sequence SEQ ID NO Alpha (α) S4 population CDR1α DRGSQS SEQ ID NO: 34 CDR2α IYSNGD SEQ ID NO: 35 CDR3α CAVIGGTDSWGKLQF SEQ ID NO: 36 variable MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVIGGTDSWGKLQFGAGTQVVVTPD SEQ ID NO: 37 Full - with TRAC constant domain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVIGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNS AVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 38 Full - with TRAC constant domain MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVIGGTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNS AVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 214 Beta (β) S4 group CDR1β LGHNA SEQ ID NO: 39 CDR2β YSLEER SEQ ID NO: 40 CDR3β CASSQEEGAVYGYTF SEQ ID NO: 41 variable MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSQEEGAVYGYTFGSGTRLTVVE SEQ ID NO: 42 Full - has TRBC1 constant domain MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSQEEGAVYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSR LRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 43 Full - has TRBC2 constant domain MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSQEEGAVYGYTFGSGTRLTVVEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 44 Full - has TRBC2 constant domain MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSQEEGAVYGYTFGSGTRLTVVEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 215 Alpha (α) S1 IFNg enriched population CDR1α NSASQS SEQ ID NO: 45 CDR2α VYSSGN SEQ ID NO: 46 CDR3α CVVPRGLSTDSWGKLQF SEQ ID NO: 47 variable MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVPRGLSTDSWGKLQFGAGTQVVVTPD SEQ ID NO: 48 Full - with TRAC constant domain MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVPRGLSTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 49 Full - with TRAC constant domain MISLRVLLVILWLQLSWVWSQRKEVEQDPGPFNVPEGATVAFNCTYSNSASQSFFWYRQDCRKEPKLLMSVYSSGNEDGRFTAQLNRASQYISLLIRDSKLSDSATYLCVVPRGLSTDSWGKLQFGAGTQVVVTPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAV AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 216 Beta (β) S1 IFNg enriched population CDR1β LNHNV SEQ ID NO: 50 CDR2β YYDKDF SEQ ID NO: 51 CDR3β CATSREGLAADTQYF SEQ ID NO: 52 variable MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSREGLAADTQYFPGGTRLTVLE SEQ ID NO: 53 Full - has TRBC1 constant domain MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSREGLAADTQYFPGGTRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRY CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 54 Full - has TRBC2 constant domain MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSREGLAADTQYFPGGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRY CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 55 Full - has TRBC2 constant domain MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSREGLAADTQYFPGGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRY CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 217 Patient 1 chain district amino acid sequence SEQ ID NO Alpha (α) is sequenced directly at the time of sorting CDR1α VSNAYN SEQ ID NO: 91 CDR2α GSKP SEQ ID NO: 92 CDR3α CAAPNDYKLSF SEQ ID NO: 93 variable MALQSTLGAVWLGLLLNSLWKVAESKDQVFQPSTVASSEGAVVEIFCNHSVSNAYNFFWYLHFPGCAPRLLVKGSKPSQQGRYNMTYERFSSSLLILQVREADAAVYYCAAPNDYKLSFGAGTTVTVRAN SEQ ID NO: 94 Full - with TRAC constant domain MALQSTLGAVWLGLLLNSLWKVAESKDQVFQPSTVASSEGAVVEIFFCNHSVSNAYNFFWYLHFPGCAPRLLVKGSKPSQQGRYNMTYERFSSSLLILQVREADAAVYYCAAPNDYKLSFGAGTTVTVRANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 95 Beta (β) 1 is sequenced directly at the time of sorting CDR1β SEHNR SEQ ID NO: 96 CDR2β FQNEAQ SEQ ID NO: 97 CDR3β CASSSSGLAFYEQYF SEQ ID NO: 98 variable MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSSGLAFYEQYFPGGTRLTVTE SEQ ID NO: 99 Full - has TRBC1 constant domain MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSSGLAFYEQYFGPGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRY CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 100 Full - has TRBC2 constant domain MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSSGLAFYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRY CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 101 Beta (β) 2 is sequenced directly at the time of sorting CDR1β SGHDN SEQ ID NO: 102 CDR2β FVKESK SEQ ID NO: 103 CDR3β CASSQLSGRDSYEQYF SEQ ID NO: 104 variable MVSRLLSLVSLCLLGAKHIEAGVTQFPSHSVIEKGQTVTLRCDPISGHDNLYWYRRVMGKEIKFLLHFVKESKQDESGMPNNRFLAERTGGTYSTLKVQPAELEDSGVYFCASSQLSGRDSYEQYFPGGTRLTVTE SEQ ID NO: 105 Full - has TRBC1 constant domain MVSRLLSLVSLCLLGAKHIEAGVTQFPSHSVIEKGQTVTLRCDPISGHDNLYWYRRVMGKEIKFLLHFVKESKQDESGMPNNRFLAERTGGTYSTLKVQPAELEDSGVYFCASSQLSGRDSYEQYFGPGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 106 Full - has TRBC2 constant domain MVSRLLSLVSLCLLGAKHIEAGVTQFPSHSVIEKGQTVTLRCDPISGHDNLYWYRRVMGKEIKFLLHFVKESKQDESGMPNNRFLAERTGGTYSTLKVQPAELEDSGVYFCASSQLSGRDSYEQYFPGGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 107 Alpha (α) 1 growth community CDR1α VSGNPY SEQ ID NO: 108 CDR2α YITGDNLV SEQ ID NO: 109 CDR3α CAVRDGGATNKLIF SEQ ID NO: 110 variable MASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVKCTYSVSGNPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFCAVRDGGATNKLIFGTGTLLAVQPN SEQ ID NO: 111 Full - with TRAC constant domain MASAPISMLAMLFTLSGLRAQSVAQPEDQVNVAEGNPLTVKCTYSVSGNPYLFWYVQYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSQTSFHLKKPSALVSDSALYFCAVRDGGATNKLIFGTGTLLAVQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFAC ANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 112 Alpha (α) 2 Growth Community CDR1α NIATNDY SEQ ID NO: 113 CDR2α ikB SEQ ID NO: 114 CDR3α CLVGGYTGGFKTIF SEQ ID NO: 115 variable MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSSTLSLPRVSLSDTAVYYCLVGGYTGGFKTIFGAGTRLFVKAN SEQ ID NO: 116 Full - with TRAC constant domain MRQVARVIVFLTLSTLSLAKTTQPISMDSYEGQEVNITCSHNNIATNDYITWYQQFPSQGPRFIIQGYKTKVTNEVASLFIPADRKSSTLSLPRVSLSDTAVYYCLVGGYTGGFKTIFGAGTRLFVKANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAF NNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 117 Beta (β) growth community CDR1β MNHEY SEQ ID NO: 118 CDR2β SMNVEV SEQ ID NO: 119 CDR3β CASSTLGGELFF SEQ ID NO: 120 variable MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSTLGGELFFGEGSRLTVLE SEQ ID NO: 121 Full - has TRBC1 constant domain MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSTLGGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 122 Full - has TRBC2 constant domain MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSTLGGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 123 Patient 2 chain district amino acid sequence SEQ ID NO Alpha (α) 1 CDR1α SSVSVY SEQ ID NO: 124 CDR2α YLSGSTLV SEQ ID NO: 125 CDR3α CAVTLLSIEPSAGGYQKVTF SEQ ID NO: 126 variable MLLLLVPAFQVIFTLGGTRAQSVTQLDSQVPVFEEAPVELRCNYSSSVSVYLFWYVQYPNQGLQLLLKYLSGSTLVESINGFEAEFNKSQTSFHLRKPSVHISDTAEYFCAVTLLSIEPSAGGYQKVTFGIGTKLQVIPN SEQ ID NO: 127 Full - with TRAC constant domain MLLLLVPAFQVIFTLGGTRAQSVTQLDSQVPVFEEAPVELRCNYSSSVSVYLFWYVQYPNQGLQLLLKYLSGSTLVESINGFEAEFNKSQTSFHLRKPSVHISDTAEYFCAVTLLSIEPSAGGYQKVTFGIGTKLQVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAV AWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 128 Alpha (α) 2 CDR1α DSASNY SEQ ID NO: 129 CDR2α IRSNVGE SEQ ID NO: 130 CDR3α CAATSRDDMRF SEQ ID NO: 131 variable MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAATSRDDMRFGAGTRLTVKPN SEQ ID NO: 132 Full - with TRAC constant domain MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAATSRDDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNN SIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 133 Beta (β) 1 CDR1β SEHNR SEQ ID NO: 134 CDR2β FQNEAQ SEQ ID NO: 135 CDR3β CASSLEGRAMPRDSHQETQYF SEQ ID NO: 136 variable MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLEGRAMPRDSHQETQYFPGGTRLLVLE SEQ ID NO: 137 Full - has TRBC1 constant domain MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLEGRAMPRDSHQETQYFPGGTRLLVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPAL NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 138 Full - has TRBC2 constant domain MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLEGRAMPRDSHQETQYFPGGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPAL NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 139 Beta (β) 2 CDR1β LNHNV SEQ ID NO: 140 CDR2β YYDKDF SEQ ID NO: 141 CDR3β CATSWGLNEQYF SEQ ID NO: 142 variable MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSWGLNEQYFPGGTRLTVTE SEQ ID NO: 143 Full - has TRBC1 constant domain MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSWGLNEQYFGPGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 144 Full - has TRBC2 constant domain MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSWGLNEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 145 Patient 3 chain district amino acid sequence SEQ ID NO Alpha (α) 1 CDR1α TRDTTYY SEQ ID NO: 146 CDR2α RNSFDEQN SEQ ID NO: 147 CDR3α CALPDKVIF SEQ ID NO: 148 variable MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALPDKVIFGPGTSLSVIPN SEQ ID NO: 149 Full - with TRAC constant domain MLTASLLRAVIASICVVSSMAQKVTQAQTEISVVEKEDVTLDCVYETRDTTYYLFWYKQPPSGELVFLIRRNSFDEQNEISGRYSWNFQKSTSSFNFTITASQVVDSAVYFCALPDKVIFGPGTSLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSI IPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 150 Alpha (α) 2 CDR1α SIFNT SEQ ID NO: 151 CDR2α LYKAGEL SEQ ID NO: 152 CDR3α CAGLYATNKLIF SEQ ID NO: 153 variable MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQEPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGLYATNKLIFGTGTLLAVQPN SEQ ID NO: 154 Full - with TRAC constant domain MLLEHLLIILWMQLTWVSGQQLNQSPQSMFIQEGEDVSMNCTSSSIFNTWLWYKQEPGEGPVLLIALYKAGELTSNGRLTAQFGITRKDSFLNISASIPSDVGIYFCAGLYATNKLIFGTGTLLAVQPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANA FNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 155 Beta (β) CDR1β SGDLS SEQ ID NO: 156 CDR2β YYNGEE SEQ ID NO: 157 CDR3β CASSVSAGSGELFF SEQ ID NO: 158 variable MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVSAGSTGELFFGEGSRLTVLE SEQ ID NO: 159 Full - has TRBC1 constant domain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVSAGSTGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 160 Full - has TRBC2 constant domain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVSAGSTGELFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG SEQ ID NO: 161

Therefore, the present invention provides isolated polypeptides comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1-55, 91-161, 182-191, 194-203 and 214-217, which Fragments, variants and homologs.

In one aspect, the invention provides a TCR comprising a TCR alpha chain sequence selected from the group consisting of the HD12-HD15 alpha chain sequences of Table 1, and a TCR selected from the group consisting of the HD12-HD15 beta chain sequences of Table 1 β chain sequence.

In one aspect, the invention provides a TCR comprising a TCR alpha chain sequence selected from the group consisting of Patient 1, Patient 2, or Patient 3 alpha chain sequences of Table 1, and a TCR alpha chain sequence selected from the group consisting of Patient 1, Patient 2, or Patient 3 of Table 1. TCR β chain sequence of the group consisting of patient 3 β chain sequences.

In one aspect, the invention provides a TCR comprising a TCR alpha chain sequence selected from the group consisting of HD12, HD13, HD14, HD15, Patient 1, Patient 2, or Patient 3 alpha chain sequences of Table 1, and a TCR alpha chain sequence selected from the group consisting of Table 1 The TCR β chain sequence of the group consisting of 1’s HD12, HD13, HD14, HD15, Patient 1, Patient 2 or Patient 3 β chain sequences.

In alternative embodiments, the sequence of the full TCR beta chain mentioned in Table 1 can be replaced by the corresponding sequence below. Donor: HD12 Beta (β) Full - has TRBC1 constant domain MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHLYFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIRSTKLEDSAMYFCASSEAGLSYEQYFPGGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLR VSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 222

In some embodiments, SEQ ID NO: 10 may be replaced by SEQ ID NO: 222. Donor: HD13 Beta (β) Full - has TRBC1 constant domain MASLLFFCGAFYLLGTGSMDADVTQTPRNRITKTGKRIMLECSQTKGHDRMYWYRQDPLGLRLIYYSFDVKDINKGEISDGYSVSRQAQAKFSLSLESAIPNQTALYFCATGQATQETQYFPGGTRLLVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCL SSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 223

In some embodiments, SEQ ID NO: 21 can be replaced by SEQ ID NO: 223. Donor: HD14 Beta (β) Full - has TRBC1 constant domain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASGRGDTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 224

In some embodiments, SEQ ID NO: 32 can be replaced by SEQ ID NO: 224. Donor: HD15 Beta (β) S4 group Full - has TRBC1 constant domain MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKSLKCEQHLGHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFLHLHTLQPEDSALYLCASSQEEGAVYGYTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSR LRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 225 Beta (β) S1 IFNg enriched population Full - has TRBC1 constant domain MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSREGLAADTQYFPGGTRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRY CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 226

In some embodiments, SEQ ID NO: 43 can be replaced by SEQ ID NO: 225.

In some embodiments, SEQ ID NO: 54 can be replaced by SEQ ID NO: 226. Patient 1 Beta (β) 1 is sequenced directly at the time of sorting Full - has TRBC1 constant domain MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSSGLAFYEQYFGPGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRY CLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 227 Beta (β) 2 is sequenced directly at the time of sorting Full - has TRBC1 constant domain MVSRLLSLVSLCLLGAKHIEAGVTQFPSHSVIEKGQTVTLRCDPISGHDNLYWYRRVMGKEIKFLLHFVKESKQDESGMPNNRFLAERTGGTYSTLKVQPAELEDSGVYFCASSQLSGRDSYEQYFGPGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 228 Alpha (α) 2 Growth Community Full - has TRBC1 constant domain MGPQLLGYVVLCLLGAGPLEAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASSTLGGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 229

In some embodiments, SEQ ID NO: 100 can be replaced by SEQ ID NO: 227.

In some embodiments, SEQ ID NO: 106 can be replaced by SEQ ID NO: 228.

In some embodiments, SEQ ID NO: 122 can be replaced by SEQ ID NO: 229. Patient 2 Beta (β) 1 Full - has TRBC1 constant domain MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLEGRAMPRDSHQETQYFPGGTRLLVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPAL NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 230 Beta (β) 2 Full - has TRBC1 constant domain MGPGLLHWMALCLLGTGHGDAMVIQNPRYQVTQFGKPVTLSCSQTLNHNVMYWYQQKSSQAPKLLFHYYDKDFNNEADTPDNFQSRRPNTSFCFLDIRSPGLGDAAMYLCATSWGLNEQYFGPGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 231

In some embodiments, SEQ ID NO: 138 may be replaced with SEQ ID NO: 230.

In some embodiments, SEQ ID NO: 144 can be replaced by SEQ ID NO: 231. Patient 3 Beta (β) Full - has TRBC1 constant domain MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVSAGSTGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF SEQ ID NO: 232

In some embodiments, SEQ ID NO: 160 may be replaced by SEQ ID NO: 232.

Reduced mismatch and improved TCR performance The TCR of the present invention can be expressed in T cells to change the antigen specificity of T cells. TCR-transduced T cells can express at least two TCR α chains and two TCR β chains. While endogenous TCR alpha/beta chains form self-tolerant receptors, introduced TCR alpha/beta chains form receptors with defined specificity for a given target antigen.

However, TCR gene therapy requires sufficient expression of the transferred TCR. The transferred TCR can be diluted by the presence of endogenous TCR, resulting in a suboptimal representation of tumor-specific TCR. Furthermore, mismatches between endogenous and introduced chains can occur to form novel receptors that may exhibit unexpected specificity for self-antigens and cause autoimmune damage when transferred into patients.

Therefore, several strategies have been developed to reduce the risk of mismatching between endogenous and introduced TCR chains. Mutation of the TCR α/β interface is one strategy currently used to reduce undesirable mismatches. For example, the introduction of cysteine in the constant domains of the alpha and beta chains allows the formation of disulfide bonds and enhances pairing of the introduced chains while reducing mispairing with the wild-type chain.

Therefore, the TCR of the invention may contain one or more mutations at the α/β chain interface such that when the α and β chains are expressed in T cells, the mismatch between these chains and the endogenous TCR α and β chains The frequency is reduced. In one embodiment, one or more mutations introduce a cysteine residue into the constant region domain of each of the alpha chain and the beta chain, wherein the cysteine residue can be present in the alpha chain and the beta chain. disulfide bonds are formed between them.

This modification of TCR is described in, for example, Boulter, J.M et al. (2003) Protein Engineering 16: 707-711 and Kuball, L. et al. (2007) Blood 109: 2331-8.

In one embodiment, the one or more mutations are at amino acid positions selected from those disclosed in Table 1 of Boulter, JM et al. (2003) Protein Engineering 16: 707-711. In one embodiment, one or more mutations replace one or more of the following amino acids with cysteine: TRAC residues TRBC residues Threonine 48 Serine 57 Threonine 45 Serine 77 Serine 61 Serine 57 Leucine 50 Serine 57 Tyrosine 10 Serine 17 Serine 15 Valine 13 Serine 15 Glutamate 15 Threonine 45 Aspartate 59 Leucine 12 Serine 17 Serine 61 Arginine 79 Leucine 12 Phenylalanine 14 Valine 22 Phenylalanine 14 Tyrosine 43 Leucine 63

In one embodiment, the TCR contains one or more of the following group of mutations: (a) Replace threonine at position 48 of the TCR α C gene with cysteine; and/or replace serine at position 57 of the TCR β C gene with cysteine; (b) Replace threonine at position 45 of the TCR α C gene with cysteine; and/or replace serine at position 77 of the TCR β C gene with cysteine; (c) Use cysteine to replace the serine at position 61 of the TCR α C gene; and/or replace the serine at position 57 of the TCR β C gene with cysteine; (d) Replace leucine at position 50 of the TCR α C gene with cysteine; and/or replace serine at position 57 of the TCR β C gene with cysteine; (e) Replace tyrosine at position 10 of the TCR α C gene with cysteine; and/or replace serine at position 17 of the TCR β C gene with cysteine; (f) Use cysteine to replace serine at position 15 of the TCR α C gene; and/or replace valine at position 13 of the TCR β C gene with cysteine; (g) Use cysteine to replace serine at position 15 of the TCR α C gene; and/or replace glutamate at position 15 of the TCR β C gene with cysteine; (h) Use cysteine to replace threonine at position 45 of the TCR α C gene; and/or replace aspartate at position 59 of the TCR β C gene with cysteine; (i) Replace leucine 12 at position 48 of the TCR α C gene with cysteine; and/or replace serine at position 17 of the TCR β C gene with cysteine; (j) Use cysteine to replace serine at position 61 of the TCR α C gene; and/or replace arginine at position 79 of the TCR β C gene with cysteine; (k) Use cysteine to replace leucine at position 12 of the TCR α C gene; and/or replace phenylalanine at position 14 of the TCR β C gene with cysteine; (l) Use cysteine to replace valine at position 22 of the TCR α C gene; and/or replace phenylalanine at position 14 of the TCR β C gene with cysteine; and/or (m) Replace tyrosine at position 43 of the TCR α C gene with cysteine; and/or replace leucine at position 63 of the TCR β C gene with cysteine.

In a preferred embodiment, the TCR comprises replacing threonine at position 48 of the TCR α C gene with cysteine; and/or replacing serine at position 57 of the TCR β C gene with cysteine.

Another strategy to reduce mismatches relies on the introduction of polynucleotide sequences encoding siRNA, added to genes encoding tumor-specific TCR alpha and or beta chains, and designed to limit the expression of endogenous TCR genes (Okamoto S. Cancer research 69, 9003-9011, 2009).

Thus, a vector or polynucleotide encoding a TCR of the invention may contain one or more siRNAs or other agents designed to limit or eliminate expression of the endogenous TCR gene.

Artificial nucleases (such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or CRISPR/Cas systems) can also be combined, which are designed to target endogenous genes (such as TCR genes (TRAC and or TRBC)) to obtain permanent disruption of the endogenous TCR alpha and/or beta chain genes, thereby allowing full expression of tumor-specific TCRs and thus reducing or eliminating the risk of TCR mismatches. This approach, known as TCR gene editing, proved superior to in vitro and in vivo TCR gene transfer (Provasi E., Genovese P., Nature Medicine May;18(5):807-15; 2012; Mastaglio S. et al. (2017) Blood 130: 606-618).

Therefore, the TCRs of the present invention can be used to edit T cell specificity through TCR disruption and genetic addition of tumor-specific TCRs.

In addition, genome editing technology allows the targeted integration of an expression cassette containing a polynucleotide encoding the TCR of the invention and optionally one or more promoter regions and/or into endogenous genes destroyed by artificial nucleases. or other expression control sequences (Lombardo A., Nature biotechnology 25, 1298-1306; 2007).

Therefore, the TCR of the present invention can be used to target and integrate the polynucleotide encoding the TCR of the present invention into the gene body region to edit T cell specificity. Integration can be targeted by artificial nucleases.

Thus, cells such as T cells can be genetically engineered to contain the TCR of the invention. Additionally, cells such as T cells can be genetically edited by gene disruption, such as TRAC and/or TRBC disruption, such as by, for example, CRISPR/Cas9, or by, for example, targeted integration of expression cassettes into source genes (e.g., endogenous genes involved in antigen specificity, persistence, expansion, activity, resistance to exhaustion/senescence/inhibitory signals, homing ability, or other T cell functions).

Another strategy developed to increase the performance of metastatic TCRs and reduce TCR mismatches consists of "murineization", which replaces the human TCR alpha and TCR beta constant regions (such as TRAC, TRBC1 and TRBC2) with their murine counterparts district). Murinization of TCR constant regions is described, for example, in Sommermeyer and Uckert J Immunol; 2010 (184:6223-6231). Therefore, the TCR of the present invention can be murine-derived.

Isolated polynucleotide The present invention relates to isolated polynucleotides encoding a TCR of the invention or a portion thereof (eg, alpha chain and/or beta chain, variable domain, or portion thereof).

The isolated polynucleotide can be double-stranded or single-stranded, and can be RNA or DNA.

Skilled artisans will understand that due to the degeneracy of the genetic code, a variety of different polynucleotides may encode the same polypeptide. In addition, it is understood that skilled artisans can use routine techniques to make nucleotide substitutions, additions or deletions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the characteristics of any particular host organism in which the polypeptides of the invention are intended to be expressed. Codon usage.

The polynucleotides described herein may be modified by any method available in the art. Such modifications can be implemented to enhance the in vivo activity or longevity of the polynucleotides of the invention.

Polynucleotides (eg, DNA polynucleotides) can be produced recombinantly, synthetically, or by any means available to those skilled in the art. They can also be selectively bred using standard techniques.

Longer polynucleotides will typically be produced using recombinant means, such as using polymerase chain reaction (PCR) selection cloning techniques. This will involve preparing a pair of primers (e.g., of about 15 to 30 nucleotides) flanking the target sequence desired for cloning, contacting the primers with mRNA or cDNA obtained from an animal or human cell, and inducing amplification of the desired region. A polymerase chain reaction is performed under the conditions, the amplified fragments are separated (for example, by purifying the reaction mixture using agarose gel), and the amplified DNA is recovered. Primers can be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate vector.

Examples of nucleotide sequences encoding TCRs according to the present invention are provided in Table 2. Table 2 Donor chain Nucleotide sequence SEQ ID NO HD12 α ( with TRAC) ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCTAGGACCTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATGTTCGTGCAGGAAAAGGAGGCTGTGACTCTGGACTGCACATATGACACCAGTGATCAAAGTTATGGTCTATTCTGGTACAAGCAGCCCAGCAGTGGGGAAATGATTTTTCTTATTTATCAGGGGTCTTATGACGAGCAAAATGCAACAGAAGGTCGCTACTCATTGAATTTC CAGAAGGCAAGAAAATCCGCCAACCTTGTCATCTCCGCTTCACAACTGGGGGACTCAGCAATGTATTTCTGTGCAATGAGAACGGGAGGAGGTGCTGACGGACTCACCTTTGGCAAAGGGACTCATCTAATCATCCAGCCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGA TGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCC GGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC SEQ ID NO: 56 α ( with TRAC) ATGAGCCTGTCTAGCCTGCTGAAGGTGGTCACAGCCAGCCTGTGGCTCGGACCTGGAATCGCCCAGAAGATCACCCAGACACAGCCCGGCATGTTCGTGCAAGAGAAAGAAGCCGTGACACTGGACTGCACCTACGACACCAGCGATCAGAGCTACGGCCTGTTCTGGTACAAGCAGCCTAGCAGCGGCGAGATGATCTTCCTGATCTACCAGGGCAGCTACGACGAGCAGAATGCCACCGAGGGCAGATACAGC CTGAACTTCCAGAAGGCCCGGAAGTCCGCCAACCTGGTCATTTCTGCTTCTCAGCTGGGCGACAGCGCCATGTACTTTTGCGCCATGAGAACAGCCGGCGGAGCCGATGGACTGACATTTGGCAAGGGCACCCACCTGATCATCCAGCCTTACATTCAGAACCCCGATCCTGCCGTGTACCAGCTGAGGGATAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCGACCAACGTGTCCCAGAG CAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACACAGCATTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTCAATTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGC TGAAAGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGTCCAGC SEQ ID NO: 204 β ( with TRBC1) ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCAGGACTCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTCACACAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACTTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTCTGGTTTCCTTTTATAATAATGAAATCTCAGAGAAGTCTGAAATATTCGATGATCAATTCTCAGTTG AAAGGCCTGATGGATCAAATTTCACTCTGAAGATCCGGTCCACAAAGCTGGAGGACTCAGCCATGTACTTCTGTGCCAGCAGTGAAGCGGGACTTTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAG CTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGC AGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC SEQ ID NO: 57 β ( with TRBC2) ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGCAGGACTCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTCACACAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTAATCACTTATACTTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTCGAGTTTCTGGTTTCCTTTTATAATAATGAAATCTCAGAGAAGTCTGAAATATTCGATGATCAATTCTCAGTTG AAAGGCCTGATGGATCAAATTTCACTCTGAAGATCCGGTCCACAAAGCTGGAGGACTCAGCCATGTACTTCTGTGCCAGCAGTGAAGCGGGACTTTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAG CTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGC AGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC SEQ ID NO: 58 β ( with TRBC2) ATGGATACTTGGCTTGTGTGCTGGGCCATCTTCAGCCTGCTGAAGGCCGGACTGACAGAGCCCGAAGTGACACAGACACCCAGCCACCAAGTGACCCAGATGGGCCAAGAAGTGATCCTGCTGCGTGCCCATCAGCAACCACCTGTACTTCTACTGGTACAGACAGATCCTGGGCCAGAAAGTGGAATTCCTGTGTCCTTCTACAACAACGAGATCAGCGAGAAGTCCGAGATCTTCGACGACCAGTTCA GCGTGGAAAGACCCGACGGCAGCAACTTCACCCTGAAGATCAGAAGCACCAAGCTCGAGGACAGCGCCATGTACTTTTGCGCCTCTTCTGAAGCCGGCCTGAGCTACGAGCAGTACTTTGGCCCTGGCACCAGACTGACCGTGACCGAGGATCTGAAGAACGTGTTCCCACCTGAGGTGGCCGTGTTCGAACCTTCTGAGGCCGAGATCTCTCACACCCAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTATCCCGA TCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGTACCGATCCTCAGCCTCTGAAAGAGCAGCCCGCTCTGAACGACAGCAGATACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGTCCGAGAACGATGAGTGGACCCAGGATAGAGCCAAGCCTGTGACTCAGATCGTGTCTGCCGAA GCCTGGGGCAGAGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGTCTGCCACAATCCTGTACGAGATCCTGCTGGGCAAAGCCACTCTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGATAGCAGGGGC SEQ ID NO: 205 Donor chain Nucleotide sequence SEQ ID NO HD13 α ( with TRAC) ATGAAGTTGGTGACAAGCATTACTGTACTCCTATCTTTGGGTTATGGGTGATGCTAAGACCACACAGCCAAATTCAATGGAGAGTAACGAAGAAGAGCCTGTTCACTTGCCTTGTAACCACTCCACAATCAGTGGAACTGATTACATACATTGGTATCGACAGCTTCCCTCCCAGGGTCCAGAGTACGTGATTCATGGTCTTACAAGCAATGTGAACAACAGAATGGCCTCTCTGGCAATCGCTGAAGACAGAAAGTC CAGTACCTTGATCCTGCACCGTGCTACCTTGAGAGATGCTGCTGTGTACTACTGCATCCTGAGTACCCGGGTCTGGGCTGGGAGTTACCAACTCACTTTCGGGAAGGGGACCAAACTCTCGGTCATACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGA CAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCT CATGACGCTGCGGCTGTGGTCCAGC SEQ ID NO: 59 α ( with TRAC) ATGAAGCTGGTCACCAGCATCACCGTGCTGCTGAGCCTGGGCATTATGGGCGACGCCAAGACCACACAGCCCAACAGCATGGAAAGCAACGAAGAGGAACCCGTGCATCTGCCCTGCAACCACAGCACAATCAGCGGCACCGACTACATCCACTGGTACAGACAGCTGCCCAGCCAGGGACCTGAGTATGTGATCCACGGCCTGACCAGCAACGTGAACAACAGAATGGCCAGCCTGGCTATCGCCGAGGACAGAAAGA GCAGCACCCTGATCCTGCACAGAGCCACACTGAGAGATGCCGCCGTGTACTACTGCATCCTGAGCACAAGAGTGTGGGCCGGCAGCTACCAGCTGACATTTGGCAAGGGCACCAAGCTGAGCGTGATCCCCAACATTCAGAACCCCGATCCTGCCGTGTACCAGCTGCGGGATAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACAT CACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCTGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAAACTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGTCCGTGATCGGCTTCCGGATCCTGCTGCTGAAAGTGGCCGGCTTCA ACCTGCTGATGACCCTGAGACTGTGGTCCAGC SEQ ID NO: 206 β ( with TRBC1) ATGGCCTCCCTGCTCTTCTTCTGTGGGCCTTTTATCTCCTGGGAACAGGGTCCATGGATGCTGATGTTACCCAGACCCCAAGGAATAGGATCACAAAGACAGGAAAGAGGATTATGCTGGAATGTTCTCAGACTAAGGGTCATGATAGAATGTACTGGTATCGACAAGACCCAGGACTGGGCCTACGGTTGATCTATTACTCCTTTGATGTCAAAGATATAAACAAAGGAGAGATCTCTGATGGATACAGTGTTCCTCG ACAGGCACAGGCTAAATTCTCCCTGTCCCTAGAGTCTGCCATCCCCAACCAGACAGCTCTTTACTTCTGTGCCACCGGCCAGGCGACCCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCGTGGAGCTGAGC TGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCCGAGGCCTGGGGTAGAGCAGACTGT GGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC SEQ ID NO: 60 β ( with TRBC2) ATGGCCTCCCTGCTCTTCTTCTGTGGGCCTTTTATCTCCTGGGAACAGGGTCCATGGATGCTGATGTTACCCAGACCCCAAGGAATAGGATCACAAAGACAGGAAAGAGGATTATGCTGGAATGTTCTCAGACTAAGGGTCATGATAGAATGTACTGGTATCGACAAGACCCAGGACTGGGCCTACGGTTGATCTATTACTCCTTTGATGTCAAAGATATAAACAAAGGAGAGATCTCTGATGGATACAGTGTTCCTCG ACAGGCACAGGCTAAATTCTCCCTGTCCCTAGAGTCTGCCATCCCCAACCAGACAGCTCTTTACTTCTGTGCCACCGGCCAGGCGACCCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGC TGGTGGGTGAATGGGAAGGAGGTGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCCGAGGCCTGGGGTAGAGCAGACTGT GGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC SEQ ID NO: 61 β ( with TRBC2) ATGGCTTCTCTTCTGTTTTTCTGCGGCGCCTTCTACCTGCTCGGCACCGGATCTATGGACGCCGACGTTACCCAGACACCACGGAACAGAATCACCAAGACCGGCAAGCGGATCATGCTGGAATGCAGCCAGACCAAGGGCCACGACCGGATGTACTGGTACAGACAGGATCCAGGACTGGGCCTGAGACTGATCTACTACAGCTTCGACGTGAAGGACATCAACAAGGGCGAGATCAGCGACGGCTACAGCGTGTCAA GACAGGCCCAGGCCAAGTTCAGCCTGAGCCTGGAAAGCGCTATCCCCAACCAGACAGCCCTGTACTTTTGTGCCACCGCCAGGCCACACAAGAGACACAGTATTTCGGCCCTGGCACCAGACTGCTGGTGCTGGAAGATCTGAAGAACGTGTTCCCACCTGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAGATCTCTCACACCCAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTATCCCGATCACGTGGA ACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGTACCGATCCTCAGCCTCTGAAAGAGCAGCCCGCTCTGAACGACAGCAGATACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGATGAGTGGACCCAGGATAGAGCCAAGCCTGTGACAGATCGTGTCTGCCGAAGCCTGGGGCA GAGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGTCTGCCACAATCCTGTACGAGATCCTGCTGGGCAAAGCCACTCTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGATAGCAGGGGC SEQ ID NO: 207 Donor chain Nucleotide sequence SEQ ID NO HD14 α (TRAV12-3*01) ( with TRAC) ATGATGAAATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGGATCCTGGACCACTCAGTGTTCCAGAGGGAGCCATTGTTTCTCCAACTGCACTTACAGCAACAGTGCTTTTCAATACTTCATGTGGTACAGACAGTATTCCAGAAAAGGCCCTGAGTTGCTGATGTACACATACTCCAGTGGTAACAAAGAAGATGGAAGGTTTACAGCACAGG TCGATAAATCCAGCAAGTATATCTCCTTGTTCATCAGAGACTCACAGCCCAGTGATTCAGCCACCTACCTCTGTGCCTCAGGAGGAGGTGCTGACGGACTCACCTTTGGCAAAGGGACTCATCTAATCATCCAGCCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTG TATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGT TTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC SEQ ID NO: 62 α (TRAV12-2*01) ( with TRAC) ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGC TCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGTGATTCAGCCACCTACCTCTGTGCCTCAGGAGGAGGTGCTGACGGACTCACCTTTGGCAAAGGGACTCATCTAATCATCCAGCCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTG TATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGT TTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC SEQ ID NO: 192 α (TRAV12-2*02) ( with TRAC) ATGATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTCCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACA GCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCTCAGGAGGAGGTGCTGACGGACTCACCTTTGGCAAAGGGACTCATCTAATCATCCAGCCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATG TGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGG GTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC SEQ ID NO: 193 α TRAV12-3*01 WT ( with TRAC) ATGATGAAGTCCCTGCGGGTGCTGCTGGTCATCCTGTGGCTGCAACTGAGCTGGGTCTGGTCCCAGCAGAAAGAGGTGGAACAGGACCCTGGACCTCTGTCTGTTTCCTGAGGGCGCCATCGTGTCCCTGAACTGCACCTACAGCAACAGCGCCTTCCAGTACTTCATGTGGTACAGACAGTACAGCCGGAAGGGCCCCGAGCTGCTGATGTACACATACAGCAGCGGCAACAAAGAGGACGGCCGGTTTACAGCCCAGG TGGACAAGAGCAGCAAGTACATCTCCCTGTTCATCCGGGACAGCCAGCCTAGCGATAGCGCCACATATCTGTGTGCATCTGGCGGCGGAGCCGATGGCCTGACATTTGGAAAGGGCACCCACCTGATCATCCAGCCTTACATTCAGAACCCCGATCCTGCCGTGTACCAGCTGAGAGACAGCAAGTCCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACG TGTACATTACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACTCCGCCGTGGCCTGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACACAGCATTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAAGTGGCCGG CTTCAACCTGCTGATGACCCTGAGACTGTGGTCCAGC SEQ ID NO: 208 α TRAV12-2*01 WT ( with TRAC) ATGAAGTCCCTGAGAGTGCTGCTGGTCATCCTGTGGCTGCAGCTGTCTTGGGTCTGGTCCCAGCAGAAAGAGGTGGAACAGAACAGCGGCCCTCTGTCTGTTTCCTGAAGGCGCTATCGCCAGCCTGAACTGCACCTACAGCGATAGAGGCAGCCAGAGCTTCTTCTGGTACAGACAGTACAGCGGCAAGAGCCCCGAGCTGATCATGTTCATCTACAGCAACGGCGACAAAGAGGACGGCCGGTTTACAGCCCAGCT GAACAAGGCCAGCCAGTACGTTCCCTGCTGATCAGAGATAGCCAGCCTAGCGACAGCGCCACCTACCTTTGTGCATCTGGTGGCGGAGCCGATGGCCTGACATTTGGCAAGGGAACCCACCTGATCATCCAGCCTTACATTCAGAACCCCGATCCTGCCGTGTACCAGCTGAGAGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACG TGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAGCATTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAAGTGGCCGG CTTCAACCTGCTGATGACCCTGAGACTGTGGTCCAGC SEQ ID NO: 209 α TRAV12-2*01 mut ( with TRAC) ATGAAGTCCCTGAGAGTGCTGCTGGTCATCCTGTGGCTGCAGCTGTCTTGGGTCTGGTCCCAGCAGAAAGAGGTGGAACAGAACAGCGGCCCTCTGTCTGTTTCCTGAAGGCGCTATCGCCAGCCTGAACTGCACCTACAGCGATAGAGGCAGCCAGAGCTTCTTCTGGTACAGACAGTACAGCGGCAAGAGCCCCGAGCTGATCATGTTCATCTACAGCAACGGCGACAAAGAGGACGGCCGGTTTACAGCCCAGCT GAACAAGGCCAGCCAGTACGTTCCCTGCTGATCAGAGATAGCCAGCCTAGCGACAGCGCCACCTACCTTTGTGCATCTGGTGGCGGAGCCGATGGCCTGACATTTGGCAAGGGAACCCACCTGATCATCCAGCCTTACATTCAGAACCCCGATCCTGCCGTGTACCAGCTGAGAGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACACAGGTGTCCCAGAGCAAGGACAGCGACG TGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAGCATTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAAGTGGCCGG CTTCAACCTGCTGATGACCCTGAGACTGTGGTCCAGC SEQ ID NO: 210 α TRAV12-2*02 WT ( with TRAC) ATGATGAAGTCCCTGCGGGTGCTGCTGGTCATCCTGTGGCTGCAACTGAGCTGGGTCTGGTCCCAGCAGAAAGAGGTGGAACAGAACAGCGGCCCTCTGTCTGTTTCCTGAAGGCGCTATCGCCAGCCTGAACTGCACCTACAGCGATAGAGGCAGCCAGAGCTTCTTCTGGTACAGACAGTACAGCGGCAAGAGCCCCGAGCTGATCATGAGCATCTACAGCAACGGCGACAAAGAGGACGGCCGGTTTACAGCCCAGCT GAACAAGGCCAGCCAGTACGTTCCCTGCTGATCAGAGATAGCCAGCCTAGCGACAGCGCCACCTACCTTTGTGCATCTGGTGGCGGAGCCGATGGCCTGACATTTGGCAAGGGAACCCACCTGATCATCCAGCCTTACATTCAGAACCCCGATCCTGCCGTGTACCAGCTGAGAGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACG TGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAGCATTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAAGTGGCCGG CTTCAACCTGCTGATGACCCTGAGACTGTGGTCCAGC SEQ ID NO: 211 α TRAV12-2*02 mut ( with TRAC) ATGATGAAGTCCCTGCGGGTGCTGCTGGTCATCCTGTGGCTGCAACTGAGCTGGGTCTGGTCCCAGCAGAAAGAGGTGGAACAGAACAGCGGCCCTCTGTCTGTTTCCTGAAGGCGCTATCGCCAGCCTGAACTGCACCTACAGCGATAGAGGCAGCCAGAGCTTCTTCTGGTACAGACAGTACAGCGGCAAGAGCCCCGAGCTGATCATGAGCATCTACAGCAACGGCGACAAAGAGGACGGCCGGTTTACAGCCCAGCT GAACAAGGCCAGCCAGTACGTTCCCTGCTGATCAGAGATAGCCAGCCTAGCGACAGCGCCACCTACCTTTGTGCATCTGGTGGCGGAGCCGATGGCCTGACATTTGGCAAGGGAACCCACCTGATCATCCAGCCTTACATTCAGAACCCCGATCCTGCCGTGTACCAGCTGAGAGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACACAGGTGTCCCAGAGCAAGGACAGCGACG TGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAGCATTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAAGTGGCCGG CTTCAACCTGCTGATGACCCTGAGACTGTGGTCCAGC SEQ ID NO: 212 β ( with TRBC1) ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAACA GTTCCCTGACTTGCACTCTGAACTAAACCCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCGGGAGGGGGGACACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCGTGGAGCTGAGC TGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCCGAGGCCTGGGGTAGAGCAGACTGT GGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC SEQ ID NO: 63 β ( with TRBC2) ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAACA GTTTCCCTGACTTGCACTCTGAACTAAACCCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCGGGAGGGGGGACACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCGACCACGTGGAGCTGAGC TGGTGGGTGAATGGGAAGGAGGTGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCCGAGGCCTGGGGTAGAGCAGACTGT GGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC SEQ ID NO: 64 β TRAV12-3*01 WT ( with TRBC2) ATGGGGTTTTAGACTGCTGTGCTGCGTGGCCTTCTGTCTGCTTGGAGCTGGCCCTGTGGATAGCGGCGTTACCCAGACACCTAAGCACCTGATCACAGCCACAGGCCAGCGCGTGACCCTGAGATGTTCTCCTAGAAGCGGCGACCTGAGCGTGTACTGGTATCAGCAGTCTCTGGACCAGGGCCTGCAGTTCCTGATCCAGTACTACAACGGCGAGGAAAGAGCCAAGGGCAACATCCTGGAACGGTTTCAG CGCCCAGCAGTTCCCAGATCTGCACAGCGAGCTGAACCTGAGCAGCCTGGAACTGGGAGATAGCGCCCTGTACTTCGTGCCAGCGGCAGAGGCGATACCGAGGCCTTTTTTGGCCAAGGCACCAGACTGACCGTGGTGGAAGATCTGAAGAACGTGTTCCCACCTGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAGATCAGCCACACACAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTATC CCGATCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCGTACCGATCCTCAGCCTCTGAAAGAGCAGCCCGCTCTGAACGACAGCAGATACTGCCTGTCCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGATGAGTGGACCCAGGACAGAGCTAAGCCCGTGACAGATCGTGTCTGC CGAAGCTTTGGGGCAGAGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGTCTGCCACAATCCTGTACGAGATCCTGCTGGGCAAAGCCACTCTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGATAGCAGGGGC SEQ ID NO: 213 Donor chain Nucleotide sequence SEQ ID NO HD15 S4 group α ( with TRAC) ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGC TCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGATAGGGGGAACTGACAGCTGGGGGAAATTGCAGTTTGGAGCAGGGACCCAGGTTGTGGTCACCCCAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAAGTAAGGATT CTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGT GGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC SEQ ID NO: 65 α ( with TRAC) ATGAAGTCCCTGAGAGTGCTGCTGGTCATCCTGTGGCTGCAGCTGTCTTGGGTCTGGTCCCAGCAGAAAGAGGTGGAACAGAACAGCGGCCCTCTGTCTGTTTCCTGAAGGCGCTATCGCCAGCCTGAACTGCACCTACAGCGATAGAGGCAGCCAGAGCTTCTTCTGGTACAGACAGTACAGCGGCAAGAGCCCCGAGCTGATCATGTTCATCTACAGCAACGGCGACAAAGAGGACGGCCGGTTTACAGCCCAGCT GAACAAGGCCAGCCAGTACGTCCCTGCTGATCAGAGATAGCCAGCCTAGCGACAGCGCCACCTATCTGTGTGCCGTGATCGGCGGCACAGATAGCTGGGGCAAACTCCAGTTTGGCGCTGGCACACAGGTGGTGGTCACCCCTGACATTCAGAACCCTGATCCTGCCGTGTACCAGCTGAGAGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAA GGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACAGCATTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGTCTGTGATCGGCTTCCGGATCCTGCTGCTG AAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGACTGTGGTCCAGC SEQ ID NO: 218 β ( with TRBC1) ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCGGGTGAGTTGGTCCCCATGGAAACGGGAGTTACGCAGACACCAAGACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATCTGGGTCATAACGCTATGTATTGGTACAAGCAAAGTGCTAAGAAGCCACTGGAGCTCATGTTTGTCTACAGTCTTGAAGAACGGGTTGAAAACAGTGTGCCAAGTCGCTTCTCACC TGAATGCCCCAACAGCTCTCACTTATTCCTTCACCTACACACCCTGCAGCCAGAAGACTCGGCCCTGTATCTCTGCGCCAGCAGCCAAGAAGAGGGGGCTGTCTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTTGGCCACAGGCTTCTTCCCCGACCAC GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGG TAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC SEQ ID NO: 66 β ( with TRBC2) ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCGGGTGAGTTGGTCCCCATGGAAACGGGAGTTACGCAGACACCAAGACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATCTGGGTCATAACGCTATGTATTGGTACAAGCAAAGTGCTAAGAAGCCACTGGAGCTCATGTTTGTCTACAGTCTTGAAGAACGGGTTGAAAACAGTGTGCCAAGTCGCTTCTCACC TGAATGCCCCAACAGCTCTCACTTATTCCTTCACCTACACACCCTGCAGCCAGAAGACTCGGCCCTGTATCTCTGCGCCAGCAGCCAAGAAGAGGGGGCTGTCTATGGCTACACCTTCGGTTCGGGGACCAGGTTAACCGTTGTAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCGACCAC GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGG TAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC SEQ ID NO: 67 β ( with TRBC2) ATGGGATGTAGACTTCTGTGTTGCCGTGCTGTGTCTGCTTGGAGCTGGCGAACTGGTGCCTATGGAAACCGGCGTGACCCAGACACCTAGACACCTGGTCATGGGCATGACAAACAAGAAAAGCCTGAAGTGCGAGCAGCACCTGGGCCACAATGCCATGTACTGGTACAAGCAGAGCGCCAAGAAACCCTGGAACTGATGTTCGTGTACAGCCTGGAAGAGAGGGTCGAGAACAACAGCGTGCCCAGCAG ATTCAGCCCTGAGTGCCCTAATAGCAGCCACCTGTTTCTGCATCTGCACACCCTGCAGCCTGAGGACTCTGCCCTGTATCTGTGTGCCAGCAGCCAAGAGGAAGGCGCCGTTTACGGCTACACATTTGGCAGCGGCACCAGACTGACCGTGGTGGAAGATCTGAAGAACGTGTTCCCACCTGAGGTGGCCGTGTTCGAGCCTTTCGAGGCCGAGATCAGCCACACACAGAAAGCCACACTCGTGTGTCTGGCCACCGGCT TCTATCCCGATCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGTACCGATCCTCAGGCCTCTGAAAGAGCAGCCCGCTCTGAACGACAGCAGATACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCAGAAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGATGAGTGGACCCAGGATAGAGCCAAGCCTGTGACACAGATCGT GTCTGCCGAAGCCTGGGGCAGAGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGTCTGCCACAATCCTGTACGAGATCCTGCTGGGAAAAGCCACTCTGTACGCTGTGCTGGTGTCCGCTCTGGTGCTGATGGCCATGGTCAAGCGGAAGGATAGCAGGGGC SEQ ID NO: 219 HD15 S1 IFNg enriched population α ( with TRAC) ATGATATCCTTGAGAGTTTTACTGGTGATCCTGTGGCTTCAGTTAAGCTGGGTTTGGAGCCAACGGAAGGAGGTGGAGCAGGATCCTGGACCCTTCAATGTTCCAGAGGGAGCCACTGTCGCTTTCAACTGTACTTACAGCAACAGTGCTTCTCAGTCTTTCTTCTGGTACAGACAGGATTGCAGGAAAGAACCTAAGTTGCTGATGTCCGTATACTCCAGTGGTAATGAAGATGGAAGGTTTACAGCACAGCTC AATAGAGCCAGCCAGTATATTTCCCTGCTCATCAGAGACTCCAAGCTCAGTGATTCAGCCACCTACCTCTGTGTGGTGCCCCGGGGGCTTTTCAACTGACAGCTGGGGGAAATTGCAGTTTGGAGCAGGGACCCAGGTTGTGGTCACCCCAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAA GGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTG AAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC SEQ ID NO: 68 α ( with TRAC) ATGATCAGCCTGAGAGTGCTGCTGGTCATCCTGTGGCTGCAGCTGTCTTGGGTCTGGTCCCAGCGGAAAGAGGTGGAACAGGACCCCGGACCTTTCAATGTGCCTGAAGGCGCCACCGTGGCCTTCAACTGCACCTACAGCAATAGCGCCAGCCAGAGCTTCTTCTGGTACAGACAGGACTGCCGGAAAGAACCCAAGCTGCTGATGAGCGTGTACAGCAGCGGCAACGAGGACGGCAGATTCACAGCCCAGCTGA ACAGAGCCAGCCAGTACATCAGCCTGCTGATCCGGGATAGCAAGCTGAGCGATAGCGCCACCTACCTGTGCGTGGTGCCTAGAGGCCTGAGCACAGATTCTTGGGGCAAGCTGCAGTTCGGAGCCGGAACACAGGTGGTGGTCACCCCTGACATTCAGAACCCTGATCCTGCCGTGTACCAGCTGAGAGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCC AGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGATTTCGCCTGCGCCAACGCCTTCAACACAGCATTATCCCCGAGGACACATTCTTCCCAAGTCCTGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTG CTGCTGAAAGTGGCCGGCTTCAACCTGCTCATGACCCTGAGACTGTGGTCCAGC SEQ ID NO: 220 β ( with TRBC1) ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATGGGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACCCAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTAACAATGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGA ACACTTCTTTCTGCTTCTTGACATCCGCTCACCAGGCCTGGGGGACGCAGCCATGTACCTGTGTGCCACCAGCAGGGAGGGGCTAGCGGCAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGC TGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCCGAGGCCTGGGGTAGAGCAGACTGT GGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC SEQ ID NO: 69 β ( with TRBC2) ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATGGGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACCCAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTAACAATGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGA ACACTTCTTTCTGCTTCTTGACATCCGCTCACCAGGCCTGGGGGACGCAGCCATGTACCTGTGTGCCACCAGCAGGGAGGGGCTAGCGGCAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGC TGGTGGGTGAATGGGAAGGAGGTGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCCGAGGCCTGGGGTAGAGCAGACTGT GGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC SEQ ID NO: 70 β ( with TRBC2) ATGGGACCTGGACTTCTTCATTGGATGGCCCTGTGTCTGCTCGGCACAGGACATGGCGACGCTATGGTCATTCAGAACCCCAGATACCAAGTGACCCAGTTCGGCAAGCCCGTGACACTGAGCTGTAGCCAGACACTGAACCACAACGTGATGTACTGGTATCAGCAGAAGTCCTCTCAGGCCCCTAAGCTGCTGTTCCACTACTACGACAAGGACTTCAACAACGAGGCCGACACACCCGACAACTTCCAGAGCAGAAGG CCCAATACCAGCTTCTGCTTCCTGGACATCAGAAGCCCTGGCCTGGGAGATGCCGCCATGTATCTGTGTGCCACCAGCAGAGAAGGCCTGGCCGCCGATACACAGTATTTCGGCCCTGGCACCAGACTGACCGTGCTCGAGGATCTGAAGAACGTGTTCCCACCTGAGGTGGCCGTGTTCGAGCCTTCTGAGGCCGAGATCAGCCACACACAGAAAGCCACACTCGTGTGTCTGGCCACCGGCTTCTATCCCGATC ACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGTACCGATCCTCAGCCTCTGAAAGAGCAGCCCGCTCTGAACGACAGCAGATACTGCCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCTCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGATGAGTGGACCCAGGATAGAGCCAAGCCTGTGACTCAGATCGTGTCTGCCGAAGC CTGGGGCAGAGCCGATTGTGGCTTTACCAGCGAGAGCTACCAGCAGGGCGTGCTGTCTGCCACAATCCTGTACGAGATCCTGCTGGGCAAAGCCACTCTGTACGCCGTGCTGGTGTCTGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGATAGCAGGGGC SEQ ID NO: 221 Donor chain Nucleotide sequence SEQ ID NO Patient 1 was sequenced directly at the time of sorting α ( with TRAC) ATGGCTTTGCAGAGCACTCTGGGGGCGGTGTGGCTAGGGCTTCTCCTCAACTCTCTCTGGAAGGTTGCAGAAAGCAAGGACCAAGTGTTTTCAGCCTTCCACAGTGGCATCTTCAGAGGGAGCTGTGGTGGAAATCTTCTGTAATCACTCTGTGTCCAATGCTTTACAACTTCTTCTGGTACCTTCACTTCCCGGGATGTGCACCAAGACTCCTTGTTAAAGGCTCAAAGCCTTCTCAGCAGGGACGATACAA CATGACCTATGAACGGTTCTCTTCATCGCTGCTCATCCTCCAGGTGCGGGAGGCAGATGCTGCTGTTTACTACTGTGCTGCCCCTAACGACTACAAGCTCAGCTTTGGAGCCGGAACCACAGTAACTGTAAGAGCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAAGTAAGGATTCTGATGTGTATA TCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAA TCTGCTCATGACGCTGCGGCTGTGGTCCAGC SEQ ID NO: 162 β1 ( with TRBC1) ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTG CAGAGAGGCCTAAGGGATCTTTTCCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCAGCGGACTAGCGTTCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTTGGCCACAGGCTTCTTCCCCGACC ACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGG GGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC SEQ ID NO: 163 β1 ( with TRBC2) ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTG CAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCAGCGGACTAGCGTTCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTTGGCCACAGGCTTCTACCCCGACC ACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGG GGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC SEQ ID NO: 164 β2 ( with TRBC1) ATGGTTTCCAGGCTTCTCAGTTTAGTGTCCCTTTGTCTCCTGGGAGCAAAGCACATAGAAGCTGGAGTTACTCAGTTCCCCAGCCACAGCGTAATAGAGAAGGGCCAGACTGTGACTCTGAGATGTGACCCAATTTCTGGACATGATAATCTTTATTGGTATCGACGTGTTATGGGAAAAGAAATAAAATTTCTGTTACATTTTGTGAAAGAGTCTAAACAGGATGAGTCCGGTATGCCCAATCGATTCTTAGCTGAAA GGACTGGAGGGACGTATTCTACTCTGAAGGTGCAGCCTGCAGAACTGGAGGATTCTGGAGTTTATTTCTGTGCCAGCAGCCAATTGTCAGGGCGCGACTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGGCCACAGGCTTCTTCCCCGACCAC GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGG TAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC SEQ ID NO: 165 β2 ( with TRBC2) ATGGTTTCCAGGCTTCTCAGTTTAGTGTCCCTTTGTCTCCTGGGAGCAAAGCACATAGAAGCTGGAGTTACTCAGTTCCCCAGCCACAGCGTAATAGAGAAGGGCCAGACTGTGACTCTGAGATGTGACCCAATTTCTGGACATGATAATCTTTATTGGTATCGACGTGTTATGGGAAAAGAAATAAAATTTCTGTTACATTTTGTGAAAGAGTCTAAACAGGATGAGTCCGGTATGCCCAATCGATTCTTAGCTGAAA GGACTGGAGGGACGTATTCTACTCTGAAGGTGCAGCCTGCAGAACTGGAGGATTCTGGAGTTTATTTCTGTGCCAGCAGCCAATTGTCAGGGCGCGACTCCTACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGGCCACAGGCTTCTACCCGACCAC GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGG TAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC SEQ ID NO: 166 Donor chain Nucleotide sequence SEQ ID NO Patient 1 growth community α1 ( with TRAC) ATGGCCTCTGCACCCATCTCGATGCTTGCGATGCTCTTCACATTGAGTGGCTGAGAGCTCAGTCAGTGGCTCAGCCGGAAGATCAGGTCAACGTTGCTGAAGGGAATCCTCTGACTGTGAAATGCACCTATTCAGTCTCTGGAAACCCTTATTCTTTTTTGGTATGTTCAATACCCCAACCGAGGCCTCCAGTTCCTTCTGAAATACATCACAGGGGATAACCTGGTTAAAGGCAGCTATGGCTTTGAAGCTGA ATTTAACAAGAGCCAAACCTCCTTCCACCTGAAGAAACCATCTGCCCTTGTGAGCGACTCCGCTTTGTACTTCTGTGCTGTGAGAGACGGTGGTGCTACAAACAACAAGCTCATCTTTGGAACTGGCACTCTGCTTGCTGTCCAGCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAAGTAAGGATT CTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGT GGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC SEQ ID NO: 167 α2 ( with TRAC) ATGAGGCAAGTGGCGAGAGTGATCGTGTTCCTGACCCTGAGTACTTTGAGCCTTGCTAAGACCACCCAGCCCATCTCCATGGACTCATATGAAGGACAAGAAGTGAACATAACCTGTAGCCACAACAACATTGCTACAAATGATTATATCACGTGGTACCAACAGTTTCCCAGCCAAGGACCACGATTTATTATTCAAGGATACAAGACAAAAGTTACAAACGAAGTGGCCTCCCTGTTTATCCCTGCCGACAGAAAGTCCAGCACT CTGAGCCTGCCCCGGGTTTCCCTGAGCGACACTGCTGTGTACTACTGCCTCGTGGGTGGTTATACTGGAGGCTTCAAAACTATCTTTGGAGCAGGAACAAGACTATTTGTTAAAGCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTA GACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGG CTGTGGTCCAGC SEQ ID NO: 168 β ( with TRBC1) ATGGGCCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTC TCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCGTACTTCTGTGCCAGCAGTACGCTTGGGGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGA GCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCCGAGGCCTGGGGTAGAGCAGACT GTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC SEQ ID NO: 169 β ( with TRBC2) ATGGGCCCCCCAGCTCCTTGGCTATGTGGTCCTTTGCCTTCTAGGAGCAGGCCCCCTGGAAGCCCAAGTGACCCAGAACCCAAGATACCTCATCACAGTGACTGGAAAGAAGTTAACAGTGACTTGTTCTCAGAATATGAACCATGAGTATATGTCCTGGTATCGACAAGACCCAGGGCTGGGCTTAAGGCAGATCTACTATTCAATGAATGTTGAGGTGACTGATAAGGGAGATGTTCCTGAAGGGTACAAAGTCTC TCGAAAAGAGAAGAGGAATTTCCCCCTGATCCTGGAGTCGCCCAGCCCCAACCAGACCTCTCGTACTTCTGTGCCAGCAGTACGCTTGGGGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGA GCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCCGAGGCCTGGGGTAGAGCAGACT GTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC SEQ ID NO: 170 Donor chain Nucleotide sequence SEQ ID NO Patient 2 α 1 ( with TRAC) ATGCTCCTGCTGCTCGTCCCAGCGTTTCCAGGTGATTTTTACCCTGGGAGGAACCAGAGCCCAGTCTGTGACCCAGCTTGACAGCCAAGTCCCTGTCTTTGAAGAAGCCCCTGTGGAGCTGAGGTGCAACTACTCATCGTCTGTTTCAGTGTATCTCTTCTGGTATGTGCAATACCCCAACCAAGGACTCCAGCTTCTCCTGAAGTATTTATCAGGATCCACCCTGGTTGAAAGCATCAACGGTTTGAGGCTGAATTTAA CAAGAGTCAAACTTCCTTCCACTTGAGGAAACCCTCAGTCCATATAAGCGACACGGCTGAGTACTTCTGTGCTGTGACCCTGCTTTCGATTGAGCCTTCGGCTGGGGGTTACCAGAAAGTTACCTTTGGAATTGGAACAAAGCTCCAAGTCATCCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAA GGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTG AAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC SEQ ID NO: 171 α 2 ( with TRAC) ATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGACTTGGTGAATGGAGAGAATGTGGAGCAGCATCCTTCAACCCTGAGTGTCCAGGAGGGAGACAGCGCTGTTATCAAGTGTACTTATTCAGACAGTGCCTCAAACTACTTCCCTTGGTATAAGCAAGAACTTGGAAAAAGACCTCAGCTTATTATAGACATTCGTTCAAATGTGGGCGAAAAGAAAGACCAACGAATTGCTGTTACATTGAACAAGACAGCC AAACATTTCTCCCTGCACATCACAGAGACCCAACCTGAAGACTCGGCTGTCTACTTCTGGTGCAGCAACCTCCCGCGATGACATGCGCTTTGGAGCAGGGACCAGACTGACAGTAAAACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGGTATATCACAGACAAAACTGTGC TAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTGACGCTGC GGCTGTGGTCCAGC SEQ ID NO: 172 β 1 ( with TRBC1) ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTG CAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCTTAGAAGGAAGGGCCATGCCCAGGGACAGCCACCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACCCAAAAGGCCACACTGGTGTGC CTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCA GATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC SEQ ID NO: 173 β 1 ( with TRBC2) ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTG CAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCTTAGAAGGAAGGGCCATGCCCAGGGACAGCCACCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACCCAAAAGGCCACACTGGTGTGC CTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCA GATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC SEQ ID NO: 174 β 2 ( with TRBC1) ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATGGGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACCCAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTAACAATGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGA ACACTTCTTTCTGCTTCTTGACATCCGCTCACCAGGCCTGGGGGACGCAGCCATGTACCTGTGTGCCACCAGCTGGGGGGCTAAACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGG GTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTT ACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC SEQ ID NO: 175 β 2 ( with TRBC2) ATGGGTCCTGGGCTTCTCCACTGGATGGCCCTTTGTCTCCTTGGAACAGGTCATGGGGATGCCATGGTCATCCAGAACCCAAGATACCAGGTTACCCAGTTTGGAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCATGTACTGGTACCAGCAGAAGTCAAGTCAGGCCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTAACAATGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGGCCGA ACACTTCTTTCTGCTTCTTGACATCCGCTCACCAGGCCTGGGGGACGCAGCCATGTACCTGTGTGCCACCAGCTGGGGGCTAAACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGG GTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTC ACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC SEQ ID NO: 176 Donor chain Nucleotide sequence SEQ ID NO Patient 3 α 1 ( with TRAC) ATGCTGACTGCCAGCCTGTTGAGGGCAGTCATAGCCTCCATCTGTGTTGTATCCAGCATGGCTCAGAAGGTAACTCAAGCGCAGACTGAAATTTCTGTGGTGGAGAAGGAGGATGTGACCTTGGACTGTGTGTATGAAACCCGTGATACTACTTATTACTTATTCTGGTACAAGCAACCACCAAGTGGAGAATTGGTTTTCCTTATTCGTCGGAACTCTTTTGATGAGCAAAATGAAATAAGTGGTCGGTATTCTTGGA ACTTCCAGAAATCCACCAGTTCCTTCAACTTCACCATCACAGCCTCACAAGTCGTGGACTCAGCAGTATACTTCTGTGCTCTGCCCGACAAGGTGATATTTGGGCCAGGGACAAGCTTATCAGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACA GACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTG CTCATGACGCTGCGGCTGTGGTCCAGC SEQ ID NO: 177 α 2 ( with TRAC) ATGCTCCTTGAACATTTATTAATAATCTTGTGGATGCAGCTGACATGGGTCAGTGGTCAACAGCTGAATCAGAGTCCTCAATCTATGTTTATCCAGGAAGGAGAAGATGTCTCCATGAACTGCACTTCTTCAAGCATATTTAACACCTGGCTATGGTACAAGCAGGAACCTGGGGAAGGTCCTGTCCTCTTGATAGCCTTATATAAGGCTGGTGAATTGACCTCAAATGGAAGACTGACTGCTCAGTTTGGTATAACCAGAAA GGACAGCTTCCTGAATATCTCAGCATCCATACCTAGTGATGTAGGCATCTACTTCTGTGCTGGGCTATATGCTACAAACAAGCTCATCTTTGGAACTGGCACTCTGCTTGCTGTCCAGCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGGTGTATATCACAGACAAAACTGT GCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTGACGCT GCGGCTGTGGTCCAGC SEQ ID NO: 178 β ( with TRBC1) ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAACA GTTTCCCTGACTTGCACTCTGAACTAAACCCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTATCGGCAGGGAGCACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCAC GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGG TAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC SEQ ID NO: 179 β ( with TRBC2) ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAACA GTTTCCCTGACTTGCACTCTGAACTAAACCCTGAGCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTATCGGCAGGGAGCACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTGGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCAC GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGG TAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC SEQ ID NO: 180

Accordingly, the present invention provides isolated polynucleotides comprising one or more nucleotides selected from the group consisting of SEQ ID NOs: 56-70, 162-180, 192, 193, 204-213 and 218-221 sequence, or having at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least Variants thereof with 98% or at least 99% sequence identity.

The present invention also provides a TCR comprising SEQ ID NOs: 56, 59, 62, 65, 68, 162, 167, 168, 171, 172, 177, 178, 192, 193, 204, 206, 208-212 , 218 and 220 and the nucleotide sequence of the group consisting of at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% , an alpha chain encoded by a variant thereof that has at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The invention also provides a TCR comprising SEQ ID Nos: 57, 58, 60, 61, 63, 64, 66, 67, 69, 70, 163, 164, 165, 166, 169, 170, 173, 174 , 175, 176, 179, 180, 205, 207, 213, 219 and 221, and the nucleotide sequence of the group consisting of at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least Beta strands encoded by variants thereof that have 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The invention further provides isolated polynucleotide sequences derived from the sequences presented in Table 2. For example, the invention provides isolated polynucleotides encoding the variable regions of TCRs according to the invention, wherein the isolated polynucleotides comprise SEQ ID Nos: 56-70, 162-180, 192, 193, 204 - a stretch of nucleotides any one of 213 and 218-221.

Variant sequences may have the addition, deletion, or substitution of one or more bases. If the change involves an addition or deletion, it occurs in threes or is balanced (i.e., for each deletion, there is one addition) so that the change does not cause a frame shift to translate the remainder of the sequence.

Some or all of the changes may be "silent" in the sense that they do not affect the sequence of the encoded protein due to the degeneracy of the genetic code.

Some or all changes may result in conservative amino acid substitutions, additions or deletions as explained above. The changes may be concentrated in one or more regions, such as the region encoding the constant region, the linker or the framework regions of the alpha or beta chain, or they may be diffuse throughout the molecule.

The variant sequence should retain the ability to encode all or part of the TCR amino acid sequence that binds to the WT1 peptide.

Codon optimization Polynucleotides used in the invention may be codon optimized. Codon optimization has been previously described in WO 1999/41397 and WO 2001/79518. Cells differ in their use of specific codons. This codon bias corresponds to the relative abundance of a specific tRNA in a cell type. Performance can be increased by changing the codons in the sequence so that it is adjusted to match the relative abundance of the corresponding tRNA. Likewise, performance can be reduced by appropriate selection of codons whose corresponding tRNAs are known to be rare in specific cell types. Therefore, additional degrees of translation control are available.

Many viruses, including HIV and other lentiviral species, use a large number of rare codons, and by changing these codons to correspond to commonly used mammalian codons, the expression of packaging components in mammalian production cells can be increased. Codon usage tables for mammalian cells and various other organisms are known in the art.

Codon optimization can also involve removal of mRNA destabilizing motifs and hidden splice sites.

carrier The invention provides vectors comprising polynucleotides described herein.

A tool that allows or facilitates the transfer of entities from one environment to another. In accordance with the present invention and by way of example, some vectors used in recombinant nucleic acid technology permit the transfer of entities, such as segments of nucleic acids, such as heterologous DNA segments, such as heterologous Cdna segments, into target cells. The vectors may be used in cells. The purpose of maintaining a heterologous nucleic acid (DNA or RNA), promoting the replication of a vector containing a segment of the nucleic acid, or promoting the expression of a protein encoded by a segment of the nucleic acid. The vector may be non-viral or viral. Recombinant Nucleic Acid Technology Examples of vectors used in include, but are not limited to, plastids, chromosomes, artificial chromosomes, and viruses. Vectors can be single-stranded or double-stranded. They can be linear and optionally the vector contains one or more homology arms. The vector may also be, for example, a naked nucleic acid (eg DNA). In its simplest form, the vector itself may be the nucleotide of interest.

Vectors used in the present invention may be, for example, plasmid or viral vectors, and may include a promoter for expressing the polynucleotide and optionally a regulator of the promoter.

Vectors containing polynucleotides used in the present invention can be introduced into cells using a variety of techniques known in the art, such as transformation, transfection, and transduction. Certain techniques are known in the industry, such as transduction with recombinant viral vectors (e.g. retrovirus, lentivirus, adenovirus, adeno-associated virus, baculovirus and herpes simplex virus vectors, Sleeping Beauty vectors); direct injection of nucleic acids and organisms Ballistics shift.

Non-viral delivery systems include, but are not limited to, DNA transfection methods. Here, transfection includes the process of delivering genes to target cells using non-viral vectors. Typical transfection methods include electroporation, DNA bioballistics, lipid-mediated transfection, dense DNA-mediated transfection, liposomes, immunoliposomes, cationic liposomes, cationic agent-mediated transfection, cationic surface CFA (Nature Biotechnology 1996 14;556) and combinations thereof.

The term "transfection" should be understood to encompass the delivery of polynucleotides to cells by viral and non-viral delivery.

Additionally, the present invention may employ gene targeting approaches, such as the delivery of DNA modifying agents.

The term "vector" includes expression vehicles, ie constructs capable of expression in vivo or in vitro/ex vivo. Expression may be controlled by vector sequences or, for example, in the case of target site insertion, by target sequences. The vector can be integrated or tethered to the DNA of the cell.

Viral delivery systems include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retroviral vectors, lentiviral vectors, and baculovirus vectors.

Retroviruses are RNA viruses that have a different life cycle than lytic viruses. In this regard, retroviruses are infectious entities that replicate via DNA intermediates. When a retrovirus infects a cell, its genome is converted into DNA form by reverse transcriptase. The DNA copies serve as templates for the production of new RNA genomes and virus-encoded proteins necessary for the assembly of infectious virus particles.

There are many retroviruses, such as murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus, RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytosis virus 29 (MC29), avian erythroblastosis virus (AEV), and all other reverse transcriptase Viridae, including the genus Lentivirus.

A detailed list of retroviruses can be found in Coffin et al. ("Retroviruses" 1997 Cold Spring Harbor Laboratory Press editors: JM Coffin, SM Hughes, HE Varmus pp. 758-763).

The genus Lentivirus also belongs to the family Retroviridae, but it can infect both dividing and non-dividing cells (Lewis et al. (1992) EMBO J. 3053-3058).

The vector may be capable of transferring a nucleotide sequence encoding a WT1-specific TCR described herein to a cell (eg, a T cell) such that the cell expresses the WT1-specific TCR. Preferably, the vector will be able to sustain high levels of expression in T cells such that the introduced TCR can successfully compete with the endogenous TCR for the limited pool of CD3 molecules.

Increasing the supply of CD3 molecules may, for example, increase TCR expression in cells that have been modified to express the TCR of the invention. Therefore, the vector of the present invention may further comprise one or more genes encoding CD3-γ, CD3-δ, CD3-ε and/or CD3-ζ. In one embodiment, the vector of the invention contains a gene encoding CD3-ζ. The vector may contain a gene encoding CD8. The vector may encode a selectable marker or suicide gene to improve the safety profile of genetically engineered cells (e.g., cells of the invention, or cells modified to express the TCR of the invention) (Bonini, Science 1997, Ciceri, Bonini Lancet Oncol. 2009, Oliveira et al., STM 2015). The genes included in the vector of the present invention can be connected through self-cleaving sequences (eg, 2A self-cleaving sequences).

Alternatively, one or more separate vectors encoding the CD3 gene may be provided for simultaneous, sequential or separate co-transfer with one or more vectors of the invention (e.g., one or more vectors encoding the TCR of the invention). cells.

cells The invention relates to cells comprising polynucleotides or vectors according to the invention.

The cells can be T cells, lymphocytes or stem cells. T cells, lymphocytes or stem cells can be selected from the following groups: CD4 cells, CD8 cells, naive T cells, memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, γ/δ T cells, natural killer (NK) cells, natural killer T (NKT) cells, interleukin-induced killer (CIK) cells, hematopoietic stem cells and pluripotent stem cells.

The type of cells can be selected to provide the desired and advantageous persistence in vivo and to provide the cells of the invention with the desired and advantageous functions and characteristics.

The cell may have been isolated from the individual.

Cells of the invention can be provided for adoptive cell transfer. The term "adoptive cell transfer" as used herein refers to the administration of a population of cells to a patient. Typically, the cells are T cells isolated from an individual and then genetically modified and cultured in vitro to express the TCR of the invention before administration to the patient.

Adoptive cell transfer can be allogeneic or autologous.

"Autologous cell transfer" is understood to mean that an initial population of cells is obtained from the same individual as the individual to whom the transduced T cell population is administered (which is then transduced according to the method of the invention, or used according to the invention). vector for transduction). Autologous transfer is advantageous because it avoids problems associated with immune incompatibility and can be used on an individual basis regardless of the availability of genetically matched donors.

"Allogeneic cell transfer" is understood to mean that a starting population of cells is obtained from an individual different from the individual to which the transduced population of cells is administered (and then transduced according to the method of the invention, or transduced with a vector according to the invention). guide). Preferably, the donor will generally be genetically matched to the ontogeny of the administered cells to minimize the risk of immune incompatibility. Alternatively, the donor may be a mismatch and unrelated to the patient.

Appropriate dosages of the transduced cell population are, for example, therapeutically and/or prophylactically effective. The dosage to be administered may depend on the individual and condition to be treated, and can be readily determined by the skilled person.

The cells can be derived from T cells isolated from the individual. T cells can be part of a mixed cell population isolated from an individual, for example, a population of peripheral blood lymphocytes (PBL). T cells within the PBL population can be activated by methods known in the art, such as using anti-CD3 and/or anti-CD28 antibodies or cell-sized beads coupled to anti-CD3 and/or anti-CD28 antibodies.

T cells can be CD4 ⁺ helper T cells or CD8 ⁺ cytotoxic T cells. The cells may be in a mixed population of CD4 ⁺ helper T cells/CD8 ⁺ cytotoxic T cells. For example, multiline activation using anti-CD3 antibodies, optionally in combination with anti-CD28 antibodies, will trigger the proliferation of both CD4 ⁺ and CD8 ⁺ T cells.

The cells can be isolated from the individual to whom the genetically modified cells are to be adoptively transferred. In this regard, the cells can be prepared by isolating T cells from an individual, optionally activating the T cells, and transferring the TCR gene to the ex vivo cells. The individual's subsequent immunotherapy can then be administered by adoptive transfer of TCR-transduced cells. As used herein, this process refers to autologous T cell transfer, ie, the administration of TCR-transduced cells to the same individual from which the T cells were originally derived.

Alternatively, T cells can be isolated from different individuals so that they are allogeneic. T cells can be isolated from the donor individual. For example, if an individual is undergoing an allogeneic hematopoietic stem cell transplant (Allo-HSCT) or a solid organ transplant or a cell transplant or stem cell therapy, the cells may be derived from the organ, tissue, or donor from which the cells were derived. The donor and the individual receiving treatment may be siblings.

Alternatively, the cells may be, or may be derived from, stem cells, such as hematopoietic stem cells (HSC). Since stem cells do not express CD3 molecules, gene transfer into HSCs will not result in TCR expression on the cell surface. However, when stem cells differentiate into lymphoid precursors that migrate to the thymus, initiating CD3 expression results in surface expression of the introduced TCR on thymocytes.

The advantage of this method is that once mature T cells are generated, they only express the introduced TCR and little or no expression of the endogenous TCR chain. This is because the expression of the introduced TCR chain inhibits the rearrangement of the endogenous TCR gene fragment, thereby Formation of functional TCR α and β genes. A further benefit is that genetically modified stem cell lines provide a continuous source of mature T cells with desired antigen specificity. Therefore, the cells can be genetically modified stem cells, preferably genetically modified hematopoietic stem cells, which upon differentiation produce T cells expressing the TCR of the invention.

Other methods known in the art may be used to reduce, limit, prevent, silence or eliminate expression of endogenous genes in cells of the invention or cells prepared by the methods of the invention.

The term "disruption" as used herein means reducing, limiting, preventing, silencing or eliminating the expression of a gene. One skilled in the art can use any method known in the art to disrupt endogenous genes, for example, any suitable method for genome editing, gene silencing, gene knockdown or gene knockout.

For example, endogenous genes can be destroyed with artificial nucleases. Artificial nucleases are, for example, artificial restriction enzymes engineered to selectively target a specific polynucleotide sequence (eg, encoding a gene of interest) and induce double-stranded breaks in that polynucleotide sequence. Typically, double-strand breaks (DSBs) are repaired by error-prone non-homologous end joining (NHEJ), thereby resulting in the formation of non-functional polynucleotide sequences that may not express the endogenous gene.

In some embodiments, the artificial nuclease is selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (eg, CRISPR/Cas9).

Methods for preparing cells (eg, T cells) of the invention may include the step of targeted integration of expression cassettes into endogenous genes (eg, endogenous TCR alpha chain genes and/or endogenous TCR beta chain genes). The term expression cassette as used herein refers to a polynucleotide sequence (eg, a DNA polynucleotide sequence) containing one or more polynucleotide sequences encoding one or more genes of interest such that the genes of interest are expressed. Endogenous sequences may facilitate expression from the expression cassette, and/or transcription control sequences within the expression cassette may facilitate expression. For example, an expression cassette may comprise a polynucleotide sequence of the invention, or a polynucleotide sequence encoding a TCR of the invention, operably linked to an expression control sequence, such as a promoter or enhancer sequence. One or more genes of interest can be located between one or more sets of restriction sites. Suitably, the restriction sites facilitate integration of the expression cassette into, for example, vector, plastid or genomic DNA (eg, host cell genomic DNA).

For example, the expression cassette of the present invention can be "cut", e.g., excised, e.g., excised, and "pasted", e.g., integrated into a second polynucleotide sequence, e.g., using one or more suitable restriction enzymes from, e.g., a vector. The first polynucleotide sequence is transferred to another.

Expression cassettes of the invention may comprise polynucleotides of the invention. The expression cassette may comprise a polynucleotide encoding one or more TCRs of the invention. The expression cassette may further comprise an antibiotic resistance gene or other selectable marker gene, which allows identification of cells that have successfully integrated the expression cassette into their DNA. The polynucleotide sequence contained in the expression cassette is operably linked to expression control sequences, such as appropriate promoter or enhancer sequences. Those skilled in the art will be able to select appropriate presentation control sequences.

The invention also encompasses cells expressing the TCR of the invention that are engineered to disrupt one or more endogenous MHC genes. Disruption of endogenous MHC genes can reduce or prevent the expression of MHC on the surface of engineered cells. Therefore, the engineered cells with reduced or no MHC expression will have limited or no ability to present antigens on their cell surfaces. Such cells are particularly advantageous for adoptive cell transfer because the cells will be non-alloreactive, eg, the cells will not present antigens that are recognized by the immune system of the individual receiving the adoptively transferred cells. Therefore, the transferred cells will not be recognized as "non-self" and adverse immune responses to the cells can be avoided. This cell is called a "universal unit" because it is suitable for adoptive transfer to a variety of different hosts regardless of HLA type.

Accordingly, the present invention provides methods for preparing non-alloreactive pluripotent T cells expressing the TCR of the present invention. The invention further provides non-alloreactive pluripotent T cells expressing the TCR of the invention.

The invention further encompasses cells engineered to disrupt another endogenous gene to modify the cell to enhance beneficial properties, characteristics or functions of the cell and/or to reduce undesirable properties, characteristics or functions. For example, by destroying endogenous cells, persistence, expansion, activity, resistance to exhaustion/senescence/inhibitory signals, homing ability, or other cellular functions can be modified. The term "modification" as used in this context refers to an alteration of one or more characteristics relative to an equivalent unmodified cell, eg, a cell in which endogenous genes have not been disrupted. . For example, the change may be an increase, enhancement, or introduction of a characteristic or function of the cell relative to an equivalent unmodified cell. Alternatively, the change may be a reduction, inhibition, or elimination of a characteristic or function of the cell relative to an equivalent unmodified cell.

The polynucleotides and vectors of the invention can be transferred to specific T cell subsets (including CD4 and or CD8, naive, memory stem T cells, central memory cells, effector memory cells or effector cells) or other cell subsets, for example Promote different in vivo persistence lengths and functions in cells of the invention.

The polynucleotides and vectors of the invention can also be transferred into T cell subsets (eg, naive, memory stem T cells, central memory cells, effector memory cells, effectors).

Depending on the background of interleukins and which are best suited for targeting specific tumor types, the polynucleotides and vectors of the invention can also be transferred to T cell subsets with different polarizations (e.g., Th0/Tc0, Th1/Tc1, Th2/Tc2 , Th17, Th22 or others).

In addition, the polynucleotides and vectors of the invention encoding the antigen-specific region of the TCR of the invention can be used in other cell subgroups (including γ/δ T cells, NK cells, NKT cells, interleukin-induced killer (CIK) cells, hematopoietic stem cells or other cells) to obtain therapeutic effects.

The present invention further provides a method for preparing cells, which method includes the step of transducing cells in vitro or ex vivo with the vector of the present invention. Various methods of transducing cells with vectors are known in the art (see, eg, Sambrook et al.).

The invention also provides methods of generating T cells expressing the TCR of the invention by inducing differentiation of stem cells comprising the polynucleotide or vector of the invention.

A population of cells can be selectively purified for cells that exhibit a particular phenotype or characteristic and from other cells that do not exhibit that phenotype or characteristic, or that exhibit that phenotype or characteristic to a lesser extent. For example, a population of cells expressing a specific marker (eg, CD3, CD4, CD8, CD25, CD127, CD152, CXCR3, or CCR4) can be purified from a starting cell population. Alternatively, or in addition, a population of cells that does not express another marker can be purified.

By "enriching" a cell population for a certain cell type, it is understood that the concentration of cells of that type increases within the population. Concentrations of other cell types may be reduced concomitantly.

Purification or enrichment can result in a population of cells that is substantially free of other cell types.

Purifying or enriching a population of cells expressing a particular marker (e.g., CD3, CD4, CD8, CD25, CD127, CD152, CXCR3 or CCR4) can be accomplished by using a method that binds to, preferably substantially specifically binds to, the marker. To achieve this through the action of agents. The agent that binds to the cell marker can be an antibody, such as an antibody that binds to CD3, CD4, CD8, CD25, CD127, CD152, CXCR3 or CCR4.

The term "antibody" refers to an intact antibody or antibody fragment capable of binding to a target of choice, and includes Fv, ScFv, F(ab') and F(ab') ₂ , monoclonal and polyclonal antibodies, engineered antibodies, including Chimeric, CDR-grafted and humanized antibodies, and artificially selected antibodies produced using phage display or alternative technologies.

In addition, substitutes for typical antibodies can also be used in the present invention, such as "avibodies", "avimers", "anticalins", and "nanobodies" and “DARPins”.

Agents that bind to a specific label can be labeled for identification using any of a variety of techniques known in the art. The agent may be inherently labeled, or may be modified by coupling a label thereto. "Coupled" is understood to mean that the agent and label are operably linked. This means that the agent and the label are linked together in a manner that allows both to perform their function (eg, bind to the label, allow fluorescent identification, or allow separation when placed in a magnetic field) with substantial unimpedance. Suitable coupling methods are well known in the art and can be readily identified by the skilled person.

Labeling may allow the labeled agent and any cells bound thereto to be purified from their environment (eg, the agent may be labeled with magnetic beads or an affinity tag (eg, avidin)), detected, or both. Detectable labels suitable for use as labels include fluorophores (such as green, cherry, cyan and orange fluorescent proteins) and peptide tags (such as His tag, Myc tag, FLAG tag and HA tag).

Many techniques for isolating populations of cells expressing specific markers are known in the art. Such techniques include magnetic bead-based separation techniques (e.g., closed-circuit magnetic bead-based separation), flow cytometry, fluorescence-activated cell sorting (FACS), affinity tag purification (e.g., using affinity columns or beads, e.g. Biotin columns separate avidin-labeled agents) and microscopy-based techniques.

Isolation can also be performed using a combination of different techniques, such as a magnetic bead-based separation step followed by sorting the resulting cell population by flow cytometry for one or more other (positive or negative) markers.

Clinical grade separation can be performed, for example, using the ^CliniMACS® system (Miltenyi). This is an example based on closed-circuit magnetic bead separation technology.

It is also contemplated that dye exclusion properties (eg, side group or rose bengal labeling) or enzymatic activity (eg, ALDH activity) may be used to enrich HSCs.

Chimeric Molecules In another aspect, the invention provides chimeric molecules comprising a TCR of the invention, a TCR encoded by a polynucleotide of the invention, or a portion thereof, coupled to a non-cellular substrate. Coupling can be covalent or non-covalent.

The non-cellular substrate can be a nanoparticle, an exosome, or any non-cellular substrate known in the industry.

The chimeric molecules of the present invention may be soluble.

In another aspect, the invention provides chimeric molecules comprising a TCR of the invention, a TCR encoded by a polynucleotide of the invention, or a portion thereof coupled to a toxin or antibody.

The toxin or antibody can be cytotoxic. The toxin can be a cytotoxic molecule or compound, such as a radioactive molecule or compound. The TCR portion of the chimeric molecule may confer the ability to recognize cells expressing the WT1 protein or peptide. Therefore, the chimeric molecule can specifically recognize and/or bind to tumor cells expressing WT1. Therefore, the chimeric molecules of the invention can provide for the delivery of cytotoxic toxins, antibodies and/or compounds targeting WT1.

WT1 related diseases WT1 is widely expressed in various blood and solid tumors, while showing restricted expression in various healthy tissues (such as gonads, uterus, kidney, mesothelium, and progenitor cells in different tissues). The inventors have identified and determined the amino acid sequence of the TCR that recognizes the WT1 peptide. Furthermore, it has been demonstrated that T cells expressing TCRs according to the invention target and kill cells presenting WT1 peptide or overexpressing WT1 protein.

Accordingly, the present invention provides methods for treating and/or preventing diseases associated with expression of WT1, which comprise the step of administering the TCR, isolated polynucleotide, vector or cell of the invention to an individual in need thereof. The present invention also provides methods for treating and/or preventing diseases associated with expression of WT1, which comprise the step of administering cells prepared by the method of the present invention to an individual in need thereof.

The present invention further provides the TCR of the present invention, the isolated polynucleotide of the present invention, the vector of the present invention, the cells of the present invention or cells prepared by the method of the present invention, which are used for treatment and/or prevention of manifestations related to WT1 disease.

The term "prevention" is intended to mean avoiding, delaying, arresting or hindering the transmission of disease. Such treatment may, for example, prevent or reduce the likelihood of developing or transmitting a disease associated with manifestations of WT1.

As used herein, "treatment" means caring for a diseased individual to ameliorate, cure, or alleviate the symptoms of the disease, or to reduce, prevent, or delay the progression of the disease.

The individual may be a human individual. The human individual may be a child. For example, the child may be less than 10 years old, less than 9 years old, less than 8 years old, less than 7 years old, less than 6 years old, less than 5 years old, less than 4 years old, less than 3 years old, or less than 2 years old. The human individual may be an infant.

Based on the expression of WT1, it may have been previously determined that the individual is in need of the TCR, isolated polynucleotide, vector or cell of the invention, or cells prepared by the method of the invention. For example, the individual may have a cell population that exhibits increased expression of WT1 relative to a healthy control cell population. WT1 expression can be determined using a variety of techniques known in the art - for example, quantitative RT-PCR can be used to determine the amount of WT1 RNA transcript, which is indicative of WT1 protein expression. Those skilled in the art will also appreciate that WT1 protein expression can be determined by performing Western blotting using commercially available WT1-specific antibodies.

The individual may also have been previously identified as having an alteration (eg, mutation or deletion) in the WT1 gene. The change can be genetic. Therefore, diseases associated with expression of WT1 may be genetic diseases. Examples of genetic disorders associated with WT1 manifestations include (but are not limited to) WAGR (Wilm's Tumor-Aniridia-Urogenital Malformation-Delay) syndrome, Denys-Drash syndrome (DDS) , Fraser syndrome (Frasier syndrome, FS), urogenital abnormality (reproductive and urinary system abnormalities) syndrome.

Individuals with genetic disorders associated with expression of WT1 may be at higher risk of developing proliferative disorders, such as cancer.

Diseases associated with expression of WT1 may be proliferative disorders.

The proliferative disorder can be a hematological malignancy or a solid tumor. Hematologic malignancies may be selected from the group consisting of: acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), lymphoblastic leukemia, myelodysplastic syndromes, lymphoma, multiple myeloma, non-Hodgkin's lymphoma and Hodgkin's lymphoma.

Solid tumors may be selected from the group consisting of: lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer, intrauterine cancer Pancreatic cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, renal cancer, soft tissue sarcoma, urothelial cancer, biliary tract cancer, glioblastoma, mesothelioma, cervical cancer and colorectal cancer cancer.

Diseases associated with manifestations of WT1 may be selected from the group consisting of: acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), lymphoblastic leukemia, myelodysplastic syndromes, lymphoma, multiple myeloma, Non-Hodgkin's lymphoma and Hodgkin's lymphoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, bile duct cancer, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, Thyroid cancer, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial cancer, biliary tract cancer, glioblastoma, mesothelioma, Cervical and colorectal cancer.

Pharmaceutical composition The TCR of the present invention, the polynucleotide of the present invention, the vector of the present invention, the cells of the present invention, the cells prepared by the method of the present invention, the chimeric molecules of the present invention and the mixed cell population of the present invention can be combined with pharmaceutically acceptable The carrier, diluent or excipient is formulated together for administration to an individual. Suitable carriers and diluents include isotonic saline solutions, such as phosphate buffered saline, and potentially human serum albumin.

The processing of cell therapy products should preferably comply with the FACT-JACIE international standard implementation of cell therapy.

Treatment In another aspect, the invention provides a method of treating and/or preventing diseases associated with expression of WT1, comprising administering to an individual in need thereof a TCR of the invention, an isolated polynucleotide of the invention, the invention The steps of the vector of the invention, the cells of the invention, the cells prepared by the method of the invention, the chimeric molecules of the invention or the mixed cell population of the invention.

The individual may be a human individual. The individual may be a non-human animal individual.

Individuals can suffer from diseases related to manifestations of WT1. Individuals may be at risk of developing diseases associated with manifestations of WT1. The individual may have been previously determined to be at risk of developing a disease associated with manifestations of WT1. Individuals may be at increased risk of developing WT1-related diseases.

Increased risk can be determined by genetic screening and/or by reviewing an individual's family history. An individual may exhibit genetic markers that indicate an increased risk of developing a disease associated with expression of WT1.

Suitably, those skilled in the art will be aware of genetic risk factors (eg, genetic markers) associated with an increased risk of developing WT1-related diseases. The skilled person can determine whether an individual is at increased risk of developing a disease associated with manifestations of WT1 using any suitable method or technique known in the art.

The individual may have previously been treated for conditions associated with manifestations of WT1. The individual may be in remission. Individuals may be resistant to chemotherapy. Individuals may be resistant to anti-WT1 therapy.

In one embodiment, a method of treating and/or preventing a disease associated with expression of WT1 includes the step of administering chemotherapy to an individual. Chemotherapy may be administered simultaneously, sequentially or separately with the TCR of the invention, the isolated polynucleotide of the invention, the vector of the invention, the cells according to the invention, the cells prepared by the methods of the invention or the chimeric molecules of the invention Invest in individuals. .

In another aspect, the invention provides a method of treating and/or preventing a disease associated with expression of WT1, comprising the step of administering a mixed cell population, wherein the mixed cell population includes a plurality of cells each expressing a different TCR of the invention. Cell populations.

In another aspect, the invention provides a mixed cell population comprising a plurality of cell populations each expressing a different TCR of the invention.

In another aspect, the present invention provides a method for preparing a mixed cell population, the mixed cell population comprising a plurality of cell populations each expressing a different TCR of the present invention, wherein the method includes in vitro or ex vivo transfection with the vector of the present invention. Steps to guide cells.

In another aspect, the invention provides a mixed cell population for the treatment and/or prevention of diseases associated with expression of WT1, wherein the mixed cell population includes a plurality of cell populations each expressing a different TCR of the invention.

For example, a mixed cell population may include a first cell population expressing a first TCR of the invention and a second cell population expressing a second TCR of the invention. For example, a mixed cell population may include a first cell population expressing a first TCR of the invention, a second cell population expressing a second TCR of the invention, a third cell population expressing a third TCR of the invention, etc. .

Each cell population of the mixed cell population may, for example, express only a single TCR of the invention. The endogenous TCR gene of the cell population in the mixed cell population can be disrupted or deleted. Expression of endogenous TCR genes of cells in a mixed cell population can be disrupted, for example, by gene editing with artificial nucleases.

In another aspect, the invention provides a TCR of the invention, an isolated polynucleotide of the invention, a vector of the invention, a cell of the invention, a cell prepared by a method of the invention, a chimeric molecule of the invention, or Use of the mixed cell population of the invention for the manufacture of medicaments for the treatment of diseases associated with expression of WT1.

Both human and veterinary treatments are within the scope of the present invention.

Unless otherwise indicated, the practice of the invention will employ conventional techniques in cell biology, molecular biology, histology, immunology, and oncology, which are within the ability of those skilled in the art. Such techniques are explained in the literature.

See, for example, Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Chapters 9, 13 and 16, John Wiley &Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley &Sons; Polak, J.M. and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. ( 1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Such plain text is incorporated herein by reference,

Various preferred features and embodiments of the invention will now be explained by means of non-limiting examples.

EXAMPLES Example 1 Materials and Methods Peptides The WT1 protein sequence previously published by Gessler et al. (Doubrovina, E. et al. Blood 120: 1633-1646 (2012)) was adopted to design peptides for stimulating and isolating WT1-specific T cells. This sequence contains 575 amino acids and includes the first 126 amino acids in the N-terminus deleted in the (exon 5+, KTS+) isoform of WT1. It consists of 141 fifteen peptides that span the entire sequence of the WT1 protein, each overlapping the next by 11 amino acids. Starting from the initial pool described in Doubrovina et al., to increase the chance of enriching WT1-specific T cells restricted to peptides processed and presented by different HLA alleles, and in particular the HLA-A*02:01 restriction element, we Use 3 different scenarios.

1. Stimulate with WT1 Pool-137: For healthy donor 12 (HD12), peripheral blood mononuclear cells (PBMC) were stimulated with a WT1 pool of 137 pentapeptides (denoted WT1 pool-137) obtained by excluding peptides 40, 41, 63, 64 to Avoid isolation of the WT1 37-45 epitope (VLDFAPPGA (SEQ ID NO: 72), an immunodominant peptide restricted to the HLA-A*02:01 allele) and the WT1 126-134 epitope (RMFPNAPYL (SEQ ID NO. : 71), an immunogen that has been described to be processed by the immunoproteasome (Jaigirdar, A. et al. J Immunother. 39(3):105-16 (2016) and presented by the HLA-A*02:01 allele peptide) T cells with specificity.

2. Stimulate with WT1-HLA-A*02:01 pool: For HD13, HD14, HD15, PBMC were stimulated with a pool of defined peptides known to be potentially restricted to the HLA-A*02:01 allele (Doubrovina, E. et al. Blood 120: 1633-1646 (2012)). Selected peptides indicated in Table 3 were pooled at a concentration of 13.6 μg/ml/peptide. The peptides were labeled according to the nomenclature described above that has been used for the WT1 pool (141 peptides) (indicated as WT1-HLA-A*02:01 pool). The peptides VLDFAPPGA (SEQ ID NO: 72) (P40-41) and RMFPNAPYL (SEQ ID NO: 71) (P63-64) are not included in the new pool.

3. Stimulation with a single peptide: PBMC of HD15 were also stimulated with a single peptide (P91) selected for its HLA restriction (probably HLA-A*02:01), its native processing, and expression on primary leukemia blasts (as reported by Doubrovina et al.).

Peptides are synthesized by PRIMM according to specifications of verified sequence, 70% purity, sterility and absence of endotoxins. Equal amounts of these peptides were mixed in a WT1 pool consisting of 137 peptides (WT1 library 137) at a concentration of 1 μg/ml/peptide. Additionally, 24 subpools were generated, each containing up to 12 peptides (4.17 μg/ml per peptide) according to a specific mapping matrix, such that each peptide was included in only two overlapping subpools, as shown in Table 4. Table 3. Peptides included in the WT1 HLA-A*02:01 pool. Peptide number peptide sequence SEQ ID NO P4 PTACPLPHFPPSLPP SEQ ID NO: 80 P7 LPPTHSPTHPPRAGT SEQ ID NO: 81 P13 LLAAILDFLLLQDPA SEQ ID NO: 82 P20 RSGPGCLQQPEQQGV SEQ ID NO: 83 P25 IWAKLGAAEASAERL SEQ ID NO: 84 P33 SDVRDLNALLPAVPS SEQ ID NO: 85 P37 GGGGGCALPVSGAAQ SEQ ID NO: 86 P91 CMTWNQMNLGATLKG SEQ ID NO: 87 P92 NQMNLGATLKGVAAG SEQ ID NO: 88 P129 TCQRKFSRSDHLKTH SEQ ID NO: 89 P131 SDHLKTHTRTHTGKT SEQ ID NO: 90 Table 4. Mapping raster strategies.

Isolation of peripheral blood mononuclear cells After signing informed consent, peripheral blood was obtained from 4 healthy donors (HD) at San Raffaele Hospital (OSR). Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque density gradient centrifugation.

Immortalized B cells Autologous B cells were isolated from PBMC of healthy donors using CD19 microbeads (Miltenyi Biotec). Cells were treated with cells carrying the BCL-6/BCL-XL transgene (Kwakkenbos, MJ et al. Nat. Med. Jan; 16(1):123-(2010)) and H/F pseudotype (Lévy, C. et al. Molecular Therapy 20 9, 1699-1712, (2012)) lentiviral vector transduction and supplemented with 10% fetal bovine serum (FBS; Carlo Erba), 1% penicillin-streptomycin (Euroclone/Lonza), 2 mM glutamine Cultured in Iscove's Modified Dulbecco's Medium (IMDM) (Euroclone/Lonza) containing acid and 50 ng/ml IL21 (Miltenyi Biotec). B cells were restimulated every 5 days by coculture with irradiated (80 Gy) CD40L-expressing mouse L-cell fibroblasts (3T3-CD40L) at a 10:1 B cell:3T3-CD40L ratio.

Cell Lines T2 and EBV-BLCL cell lines were cultured in IMDM (Euroclone/Lonza) supplemented with 1% penicillin-streptomycin, 2mM glutamine, and 10% FBS.

Leukemia cells Primary AML cell lines were obtained from the OSR Leukemia Biobank and selected based on performance of WT1 (determined by quantitative PCR) and HLA typing. In co-culture experiments, leukemia blasts were maintained in X-VIVO 15 (Euroclone/Lonza) medium supplemented with 5% HS, 1% penicillin-streptomycin, 2 mM glutamine, IL3, and G -CSF (Peprotech; both 20 ng/ml).

HLA typing is performed in OSR's HLA laboratory with high resolution for HLA-A, HLA-B, and HLA-C alleles on healthy donor samples and EBV-B lymphoblastoid cells. Line (BLCL) and primary leukemia cells were typed.

Flow cytometry using antibodies against human CD3, CD4, CD8, CD107a, interferon (IFN) gamma, tumor necrosis factor (TNF) alpha, CD33, CD117, CD34, CD14, anti-active caspase 3, and HLA-A2 FITC-, PE-, PerCP-, APC-, PE-Cy7, APC Cy7-, Pacific blue and bright purple conjugated antibodies. Cells were incubated with antibodies for 15 minutes at 4°C and washed with phosphate buffered saline (PBS) containing 1% FBS. For caspase 3 staining, cells were incubated at 4°C for 1 h. Dead cells were stained using the Zombie Aqua Fixable Viability Kit (Biolegend) according to the manufacturer's instructions. Acquire flow cytometry data using one of the following cell analyzers: BD Canto II flow cytometer, BD LSRFortessa, Cytoflex S (Beckman Coulter). Data were analyzed by Flow Jo software (Tree star Inc). For intracellular assessment of expression of interleukin secretion and degranulation markers, Fix/Perm buffer set (Biolegend) was used according to the manufacturer's instructions.

Stimulation, Isolation, and Expansion of WT1- Specific T Cells Freshly isolated PBMC were resuspended in supplemented with 5% human AB serum, 1% penicillin-streptomycin, 2 mM glutamic acid, and 1 μg/ml CD28 monoclonal antibody. (BD Biosciences) in X-VIVO 15 (Euroclone/Lonza), inoculated at a density of 10 ⁷ cells/ml, and stimulated with the following substances: 1) WT1 pool of HD12-137, 2) WT1 of HD13-HD14-HD15 -HLA-A*02:01 pool, 3) HD15 single peptide (P91).

For experiments performed with 1) and 2), antigen-specific T cells were isolated by CD137 expression after 26-30 hours. More specifically, cells were stained with PE-conjugated CD137 antibody and sorted using anti-PE microbeads (Miltenyi Biotec). CD3 cells were depleted from the CD137 fraction using CD3-microbeads (Miltenyi Biotec), irradiated with 30 Gy, and incubated in cocultures with the CD137+ fraction at a ratio of 100:1 (when possible) or at least 20:1 and 5 A density of ×10 ⁶ cells/ml was used as peptide-loaded antigen-presenting cells (APC). Use X-VIVO 15 supplemented with 5% human AB serum, 1% penicillin-streptomycin, 2 mM glutamine, 5 ng/ml IL7, 5 ng/ml IL15, and 10 ng/ml IL21 as culture medium. The medium including interleukins was changed every 2-3 days.

For experiments performed with 3), antigen-specific T cells were stimulated with the P91 epitope in RPMI (Euroclone/Lonza) supplemented with 5% human AB serum. After 6 hours, cells were harvested, washed with PBS, labeled with IFNγ capture agent, and incubated at 37°C for 45 minutes. Thereafter, cells were stained with PE-labeled antibodies against IFNγ, enriched by using anti-PE microbeads and isolated using the MACS system (Miltenyi Biotec). IFNγ-enriched T cells and IFNγ-CD3-fraction were irradiated with 30 Gy at a ratio of 100:1 and seeded at a density of 5×10 ⁶ cells/ml. Use X-VIVO 15 supplemented with 5% human AB serum, 1% penicillin-streptomycin, 2 mM glutamine, 5 ng/ml IL7, 5 ng/ml IL15, and 10 ng/ml IL21 as culture medium. The medium including interleukins was changed every 2-3 days.

Approximately 20 days later, T cells were pelleted and used for TCR sequencing analysis.

Restimulation of expanded antigen-specific T cells Cells originally stimulated using protocol 1) or 2) (described above) were restimulated with WT1-pulsed autologous APCs (PBMC CD3-depleted cells) every 7-14 days. During initial restimulation, cells were washed 2 days prior and plated in interleukin-free medium. Irradiate APCs with 30 Gy, pulse with Taichi overnight in X-VIVO 15 supplemented with 5% AB serum, or in IMDM without serum for at least 3 hours on a rotator. Pulsed APCs and effector cells were cultured in a culture medium supplemented with 5% human AB serum, 1% penicillin-streptomycin, 2 mM glutamine, 1 μg/ml CD28 monoclonal antibody, and IL7 (5 ng/ml), IL15 ( 5 ng/ml), IL21 (10 ng/ml) in X-VIVO 15 CCP culture.

Evaluate T cell responses by measuring the percentage of T cells responding to the WT1 pool-137 or WT1-HLA*A02:01 pool by co-culture of effector cells with autologous APCs (at a ratio of at least 1:1) for 6 hours. The co-culture was pulsed with the desired antigen (WT1 pool-137 or WT1-HLA*02:01 pool, WT1 subpool or unrelated peptide pool as control). Co-cultures were inoculated into cells supplemented with 5% human AB serum, 1% penicillin-streptomycin, 2 mM glutamine and supplemented with CD28 monoclonal antibody (1 μg/ml), Golgi terminator transport inhibitor (BD Biosciences; 1 μg/ml) and CD107a-FITC antibody (BD Biosciences; 4 μl/well) in X-VIVO 15 were used to evaluate degranulation. Cells were then fixed, permeabilized, and intracellularly stained to determine the percentage of IFNγ-secreting CD3+CD8+ or CD3+CD4+ cells expressing CD107a.

Mapping of immunogenic peptides WT1-specific T cells from HD12 enriched using WT1 pool-137 were seeded in separate wells and co-cultured with autologous APCs loaded with one of each WT1 subpool. WT1-specific T cells enriched for HD13 and HD14 using the WT1 HLA-A*02:01 pool were seeded in separate wells and compared with autologous cells loaded with individual peptides included in the WT1-HLA*A02:01 pool. APC co-culture. For HD15, mapping of immunogenic peptides was not performed due to reduced cellularity.

Each co-culture was seeded with an effector to target ratio of at least 1:1. T cell responses to each subpool or peptide were measured by FACS analysis as previously described. For HD12, deintegration of the mapping grid was critical to determine which shared peptide elicited a T cell response. Once immunogenic epitopes are identified, HD12, HD13, and HD14 T cells are further stimulated with APCs loaded with individual peptides.

Assessing the ability of T cells to recognize cells expressing WT1 . Different experimental methods were used to measure the ability of T cells to recognize WT1-specific and HLA-restricted target cells. For HD13 and HD14, the percentage of viable target cells expressing caspase 3 was determined. Primary leukemia blasts and T cells were incubated for 6 hours at effector to target (E:T) ratios of 10:1, 4:1, 1:1, 1:4 and 1:10. As a negative control, target cells were cultured with unrelated T lymphocytes. Cells were fixed, permeabilized using Fix/Perm buffer set (Biolegend), and stained with anti-active caspase-3-antibody (Biolegend) conjugated to Pacific Blue. Dead cells were seen when stained with the Zombie Aqua Fixable Viability Kit (Biolegend).

For the remaining donors, these functional analyzes could not be performed due to the reduced fitness of the expanded WT1-specific T cells.

Enrichment of Secreted IFNγ Cells To enrich for T cells specific for WT1 expanded from HD13, an IFNγ capture assay (Miltenyi Biotec) was performed. Briefly, T cells were stimulated with immunogenic recognition epitopes for 6 h. Cells were harvested, washed with PBS, and labeled with IFNγ-capture agent. After incubation at 37°C for 45 minutes, cells were stained with PE-labeled antibodies against IFNγ. IFNγ-secreting cells were then enriched by using anti-PE microbeads and isolated using the MACS system (Miltenyi Biotec). IFNγ-enriched T cells were expanded using the protocol described in the following paragraphs.

Expansion of WT1- specific T cells In order to further expand WT1-specific T cells from HD12, HD13, and HD14 during several restimulations with autologous APC, different protocols were used.

For HD12 and HD13, the rapid amplification protocol (REP) was used as previously described (Riddell, S. R. et al. Science 80 (1992); ME, D., LT, N., Westwood, J., JR, W. & SA , R. Cancer J. (2000)).

For HD14, allogeneic irradiated (30 Gy) feeder cells and T2-irradiated (100 Gy) cells (both Both pulsed with P13 peptide) stimulated WT1-expanded T cells (effector: T2: feed ratio = 1:5:1).

Assessment of T cell proliferative potential To determine the proliferative potential of expanded WT1-specific T cells, the IO Test Beta Mark TCR V beta profile kit (Beckman Coulter) was used according to the manufacturer's recommendations.

TCR profiling sequencing WT1-specific T cells were collected at different time points within the co-culture time frame, and RNA was extracted by using the Arcturus Pico Pure RNA extraction kit (Life Technology). Complementarity determining region (CDR) 3 sequences of WT1-specific T cells were amplified by using a modified RACE method (Ruggiero, E. et al. Nat. Commun. 6, 8081 (2015)). The samples were sequenced using an Illumina MiSeq sequencer, and CDR3 homotypes were identified using MiXCR software (Bolotin, DA et al. Nature Methods 12, 380-381 (2015)).

The lentiviral vector performs codon optimization, cysteine modification (Kuball, J. et al. (2007) Blood 109: 2331-8) and It was cloned into a lentiviral vector (LV) under a bidirectional promoter (European Patent No. 1616012). For WT1-specific T cells derived from HD14, MiXCR analysis revealed the presence of 3 possible TRAV genes in the same CDR3 region that recognizes peptide 13. Therefore, we ordered 3 different TCR constructs carrying one of the following genes: TRAV12-3*01, TRAV12-2*01, TRAV12-2*02. For the TCR carrying TRAV12-2*01 and TRAV12-2*02 genes, we tested: 1) codon-optimized, cysteine-modified forms, and 2) codon-optimized, cysteine-modified forms The form was further mutagenized to remove one N-glycosylation site in the TCR alpha constant domain (Kuball, J et al. (2009) J Exp Med 206: 463-75). Specifically, we replaced amino acid N at position 36 in the NXS/T motif with amino acid Q.

Name the HD14 source TCR as follows: TRAV 12-3*01 - Cysteine modification, codon optimization TRAV12-2*01 WT - cysteine modification, codon optimization TRAV12-2*01 mut - cysteine modification, codon optimization, mutagenesis to remove N-glycosylation site TRAV12-2*02 WT - cysteine modification, codon optimization TRAV12-2*02 mut - cysteine modification, codon optimization, mutagenesis to remove N-glycosylation site For each TCR, the alpha chain was selected in the antisense orientation under the minimal human CMV promoter and the beta chain was selected in the sense orientation under the PGK promoter. LV is packaged by an integrase-competent third-generation construct and pseudotyped with the vesicular stomatitis virus (VSV) envelope.

Vector Transduction For transduction with HD13- and HD14-TCR lentiviral vectors, T lymphocytes isolated from healthy individuals were activated following the manufacturer's instructions and analyzed using magnetic beads coupled to antibodies against CD3 and CD28. Select (ClinExVivo CD3/CD28; Invitrogen). Cells were seeded at a concentration of 1-2 × ¹⁰ cells/ml in IMDM supplemented with 1% penicillin, 1% streptomycin, 10% FBS and 5 ng/ml each of IL-7 and IL-15. culture in. For transduction, T lymphocytes were plated at 2.5 × ¹⁰ cells/ml and infected with LV for 24 h. Thereafter, T cells were cultured at 10 ⁶ cells/ml and expanded. Transduction efficiency was determined by measuring the percentage of CD3 ⁺ T cells expressing specific Vβ (HD13: no antibody available for Vβ; HD14: Vβ12).

TCR editing of T lymphocytes PBMCs from HD were sorted using magnetic beads coupled to antibodies against CD3 and CD28 (ClinExVivo CD3/CD28; Invitrogen) at a concentration of 1-2 × ¹⁰ cells/ml Inoculate in X-VIVO 15 supplemented with 1% penicillin, 1% streptomycin, 5% FBS and 5 ng/ml each of IL-7 and IL-15. Two days later, T cells were simultaneously electroporated with RNP complex (a combination of atomic TRAC or TRBC guide and Cas9 protein). On day 3, edited T lymphocytes were transduced with LV encoding HD12-, HD13-, and HD14-derived TCRs. After 6 days, the beads were removed and cells were seeded at a concentration of 1×10 ⁶ cells/ml. After 14 days, transduction efficiency was determined by measuring the percentage of CD3 ⁺ T cells expressing specific Vβ (HD12: Vβ22; HD13: no antibody available for Vβ; HD14: Vβ12). HD12-edited T cells were stained with MIX G (anti-Vβ22 antibody conjugated to FITC fluorescent dye - IO Test® Beta Mark kit, Beckman Coulter) and anti-FITC beads (Miltenyi Biotec) were used following the manufacturer's instructions. ) for sorting.

Functional assay using engineered T lymphocytes to measure the ability of HD12, HD13 and HD14 engineered T cells (by TCR gene transfer or TCR gene editing) to recognize target cells when co-cultured with: (a) for HD13 and HD14 TCR, at a 1:1 (E) effector:target (T) ratio via peptide pool (WT1 pool or unrelated) or subpools (1 and 14, both containing peptide 13, or unrelated) Pulsed T2 cells; (b) for HD12 TCR, EBV cell lines carrying the HLA-C*07:02 allele and pulsed with peptide 103 or an unrelated peptide as a control; (c) for HD14 TCR, based on HLA-A *Primary AML blasts selected for expression of the 0201 allele and WT1 antigen (at different E:T ratios, i.e. 50:1; 5:1). After 6 hours of co-culture, CD107a expression and/or IFNγ secretion on CD8 ⁺ T lymphocytes was assessed by fluorescence analysis for assays involving T2 cells or EBV cell lines and viable target cells for assays involving primary AML blasts Active Cas3 expression was used to determine the percentage of responding cells.

Results Generation of functional WT1-CTL from healthy donors. PBMC from HD12 were stimulated using a pool of 137 pentapeptides (WT1 pool-137), which differed from the original pool described by Doubrovina et al. due to the exclusion of 40, 41, 63, 64. To avoid isolating T cells specific for the WT1 37-45 epitope (VLDFAPPGA, SEQ ID NO: 72) and the WT1 126-134 epitope (RMFPNAPYL, SEQ ID NO: 71), these peptides were excluded.

Additionally, PBMC from 3 additional donors (HD13-HD15) were stimulated with a pool of WT1-HLA-A*02:01. After 26-30 hours, CD137+ T cells were sorted and co-cultured with the CD137 population, the CD3 fraction was further removed, and irradiated at 30 Gy.

Cells were repeatedly stimulated with APCs loaded with CD3-cell representatives of the peptide pool. Expansion of WT1-specific T cells was assessed over time by cytofluorometric analysis to evaluate the expression of interleukin release (IFNγ, IL-2, TNF-α) and degranulation markers (CD107a). As a negative control, cells were stimulated with a pool of peptides derived from unrelated antigens. In summary, tumor-specific T lymphocyte expansion in the CD8 fraction was observed after at least 3 stimulations with WT1 pools (Fig. 1, a, b, c, d).

For HD15, in a separate experiment, PBMC were stimulated with a single peptide (P91) and WT1-specific T cells were enriched by using IFNγ capture assay. After approximately 20 days of culture, the T cells were used for TCR sequencing analysis.

Mapping of WT1 epitopes that elicit T cell responses . To identify which pentapeptide of WT1 pool-137 elicited immune responses in HD12, a mapping grid strategy was used as previously described by Doubrovina et al. Briefly, the WT1 overlapping fifteen peptides were subdivided into 24 subpools (SPs) containing up to 12 peptides, with each of the 141 peptides described in Doubrovina et al. SP in. Enriched WT1-specific T cells were co-cultured with irradiated APCs (autologous immortalized B cells) subjected to 24 SP pulses for 6 hours, and the percentages of IFNγ secretion and CD107a expression were measured by flow cytometry. This strategy enables the detection of immunogenic peptides by deintegrating the mapped raster. For HD13 and HD14, autologous APCs were stimulated with each individual peptide included in the WT1 HLA-A*02:01 pool and used as target cells in a 6-hour co-culture experiment with WT1-specific T cells. For HD15, WT1-enriched T cells were generated when PBMC were stimulated with a WT1 HLA-A*02:01 pool, and mapping of immunogenic peptides was not performed due to reduced cellularity.

After stimulating T cells with subpools SP7 and SP21, a large amount of IFNγ secretion and a large amount of CD107a were observed (Fig. 2a, b). For HD13 and HD14 stimulated with WT1 pool HLA-A*02:01 (Fig. 2c), there was a robust immune response to P13 peptide-pulsed autologous APC (Fig. 2d, e for HD13 and HD14, respectively).

Once SPs recognized by WT1-specific T cells were identified against HD12, T lymphocytes were restimulated using CD3-depleted PBMC pulsed with the identified specific peptides after mapped grid deintegration (Fig. 3a; highlights the identification-free immune original peptide).

In a stepwise approach, to verify the immunogenic pentapeptide, a 6-hour co-culture with autologous irradiated immortalized B cells loaded with the pentamer to elicit an immune response and at least one unrelated pentamer Test T cells in the product. Increased CD107a expression and IFNγ secretion were observed with peptide 103 (Fig. 3b). The identified immunogenic pentapeptides were further used to restimulate T cells to enrich the epitope-specific population. For HD13 and HD14, T cells were restimulated with autologous APC stimulated with the recognized peptide (P13).

In silico prediction of peptide -MHC binding To predict the exact binding nonamers and their HLA constraints for each HD characterized by CD8-specific T cell expansion, the NetMHCpan 4.0 server was used (Jurtz V. et al. (2017) The Journal of Immunology). Binding predictions were only performed for peptides presented by HLA class I molecules, which have a strong preference for 9-amino acid peptides. The defined peptide will be identified as a strong binder if the rank % is below 0.5%, and as a weak binder if the rank % is between 0.5% and 2%.

In order to determine the HLA-alleles carried by HD12-HD15, the DNA of each individual was HLA typed at high resolution (for HLA-A, HLA-B, HLA-C) at the HLA and Chimerism Laboratory of Ospedale San Raffaele. allele) (Fig. 4a).

For HD12, 2 strong binders were identified: the peptide YRIHTHGVF (SEQ ID NO: 73) on the HLA-B*38:01 allele or the HLA-C*07:02 allele (strong binding) (Figure 4b) .

For HD13 and HD14, the combination of the peptide LLAAILDFL (SEQ ID NO: 74) and the HLA-A*02:01 allele was identified as a strong binder (Fig. 4c, d); in addition, for HD14, when determined by HLA-C The peptide AAILDFLLL (SEQ ID NO: 75) proved to be a strong binder when presented with the *03:03 allele (Figure 4d).

For HD15, no strong binders are predicted.

The identified WT1 peptides represent immunogenic peptides presented by different HLA alleles. To determine the HLA restriction of antigen-specific T cells identified for each HD analyzed, T lymphocytes were compared with those harboring (or not expressing) HDs. Target cells co-cultured with specific HLA class I alleles.

For HD12, a panel of EBV-BLCL carrying a single HLA allele in common with HD and pulsed with related or unrelated peptides was used as a target. The results showed that the number of cells expressing CD107a and secreting IFNγ increased when cocultured with EBV-BLCL carrying each HLA-C*07:02 allele and pulsed with WT1 P103 peptide (Fig. 5a).

For HD13 and HD14, which were previously reported to be stimulated by a pool of peptides capable of eliciting an immune response when presented via the HLA-A*02:01 allele, either by binding to a specific immunogenic epitope (P13) or not Or pulsed T2 cell co-culture to directly perform functional verification. Flow cytometry results showed that when T lymphocytes were co-cultured with P13-pulsed T2 cells, the percentage of cells expressing the CD107a marker and secreting IFNγ was greatly increased (Figure 5, b, c).

Evaluation of peptide processing by FACS . To determine the ability of WT1-expanded T cells isolated from HD13 (Fig. 6a) and HD14 (Fig. 6b) to recognize natively processed peptides and kill target cells, in co-existence with T lymphocytes After 6 hours of culture, the expression of active caspase 3 in viable primary blasts was assessed. Primary blast cells from 3 AML patients selected based on high expression of WT1 antigen and HLA typing (HLA-A*02:01) were used as target cells. Co-cultures of irrelevant effector cells with the same leukemic blasts used for HD13 and HD14 were included as controls. The results showed that both HD13 and HD14 T cells were able to recognize primary leukemic blasts, with HD14 showing greater elimination of AML blasts at different effector-to-target ratios used.

For HD12, due to the low cellularity of the cell population, no tests were performed to verify the native processing of the recognition peptide.

In summary, the ability of WT1-specific T cells derived from HD13 and HD14 to recognize target cells (leukemic blasts) expressing WT1 is indicative not only of the native processing of the recognized peptide, but also of its immunogenicity.

Immunoassay of WT1- specific T cells . To characterize the newly identified WT1-specific TCRs, flow cytometry and TCR sequencing of the TCR Vβ family were performed. FACS results indicate the prevalence of specific Vβ for HD12 and 14; for HD13, it was not possible to fully determine the dominant Vβ since the IO Test Beta Mark TCR Vβ spectrum ensures coverage of 75% of the entire spectrum of Vβ (Figure 7). For HD15 WT1-specific T cells, flow cytometric evaluation of the expressed Vβ family was not performed due to low cellularity and reduced cell adaptability. TCRαβ sequencing of WT1-specific T cells highlighted the increasing dominance of one of the two TCR chains, CDR3, in HD12, HD13, and HD14 over time (Fig. 8a-c). For HD15, significant amplification of specific TCR chains was observed both upon stimulation with the WT1 HLA-A*02:01 pool and upon stimulation with individual peptides (P91) followed by IFNγ enrichment (Fig. 8d).

Functional validation of newly cloned TCRs . TCR alpha and beta sequences isolated from HD12, HD13, HD14 and HD15 and recognizing WT1 epitopes restricted to HLA class I alleles were further modified to increase their surface expression and reduce interaction with endogenous TCRs. Chain mismatch. For the HD14 TCR, the receptor was further mutagenized to increase its functional affinity as Kuball, J et al. (2009) J Exp Med 206: 463-75. TCR genes obtained from HD12, HD13, and HD14 (all different forms were generated as described in Materials and Methods in Example 1) were cloned into bidirectional lentiviral vectors to promote the interaction of both TCR chains in transduced lymphocytes. Robust and coordinated performance. Virus production was performed on lentiviral vectors encoding the selected TCRs.

T cells from healthy individuals were transduced with lentiviral vectors previously generated from HD12, HD13 and HD14.

For HD12 TCR, activated T cells derived from 3 different healthy donors were edited as described in the Materials and Methods section (Example 1). In disrupting the endogenous TCR profile, T cells are transduced with LV encoding specific TCRα and β chain genes. After 14 days, transduction efficiency was assessed by measuring the percentage of cells expressing Vβ22. Transduced T cells were sorted based on Vβ expression on the cell surface (Fig. 9a).

By pulsing with peptide 103 (E:T ratio = 1:1) carrying the HLA-C*07:02 allele and pulsed with NYESO-1 peptide as a negative control or at reduced concentrations (from 40 µg to 0.4 pg) Co-cultures of EBV cell lines were used to assess the functional avidity of HD12-transduced edited T cells. Cytofluorescence analysis was used to determine the expression of CD107a on CD8 T cells and evaluate the ability of TCR-transduced T lymphocytes to recognize target cells. The results showed that even at a peptide concentration of 0.4 µg, HD12-transduced edited T cells specifically recognized target cells expressing the HLA allele of interest (Figure 9b).

To test HD13- and HD14-derived TCRs that recognize an HLA-A*02:01-restricted epitope (peptide 13), activated T lymphocytes isolated from a healthy individual were transduced with newly generated lentiviral vectors. The transduction efficiency of HD13 could not be measured as no antibodies exist that recognize its specific Vβ. The transduction efficiency of HD14-transduced T cells (with TCR TRAV12-2*01 WT or with TCR TRAV12-2*02 WT) was measured by assessing the percentage of Vβ expression on CD4 and CD8 T cells (Figure 11a).

In two different co-culture experiments, T cells transduced with HD13- and HD14-derived TCRs were tested for functional avidity. In the first experiment, HD13 TCR-transferred T cells and HD14 TCR-transferred T cells (those carrying the TRAV12-2*01 WT gene or TRAV12-2*02 WT gene) were compared with cells pulsed with a WT1 pool or an unrelated pool as a control. T2 cell co-culture. In the second experiment, HD13 TCR-transferred T cells and HD14 TCR-transferred T cells (carrying the TRAV12-2*01 WT gene) were compared with subpools 1 and 14 (both containing peptide 13) and subpool 6 (as negative Control) pulsed T2 cells co-culture. CD107a expression and/or IFNγ secretion on CD8 T cells was measured as readout by cytofluorescence analysis. Specific recognition of target cells pulsed by pool WT1 in T cells transduced with HD13 and HD14-derived TCRs was observed (Figure 10a and Figure 11b) and specific recognition of target cells pulsed by subpools 1 and 14 (Figure 10b and Figure 10b). 11c). Additionally, HD14-derived TCRs (carrying the TRAV12-2*02 WT gene or TRAV12-2*02 mut gene) were used in TCR editing methods in T cells from a healthy donor. After 14 days, transduction efficiency was assessed by measuring the percentage of cells expressing Vβ12 (Figure 12a).

To determine the ability of WT1-edited T cells expressing HD14-derived TCR (TRAV12-2*02 WT gene or TRAV12-2*02 mut gene) to kill primary leukemia blasts, after co-culture with T lymphocytes for 6 hours, Assess the expression of active caspase 3 in target cells. As controls, co-cultures of irrelevant effector cells with leukemic blasts and target cells cultured in the absence of effectors were included. The results showed that HD14-edited T cells were able to recognize primary leukemic blasts at the different effector-to-target ratios used (Figure 12b).

Discussing the possibility of redirecting the specificity of T cells against antigens expressed by tumor cells through genetic manipulation opens up a new therapeutic window for cancer immunotherapy. Specifically, the recent series of impressive clinical results using CAR-redirected T cells (June et al. (2015) Science Translational Medicine 280-287) have significantly increased the awareness of the scientific community, patient associations, pharmaceuticals, and the public. expect. Full utilization of this strategy depends largely on the identification of receptors specific for relevant tumor antigens. Ideally, tumor antigens must be molecules differentially expressed by tumor cells and healthy tissue that are highly immunogenic and may be involved in the development and/or progression of cancer. WT1 is an attractive target for cancer immunotherapy and ranks first on the National Cancer Institute's list of 75 cancer antigens as a priority (Cheever (2009) Clin. Cancer Res. 15: 5323-5337) . WT1 is overexpressed by cancer cells 10 to 1000 times more than healthy tissue (Inoue (1997) Blood 89: 1405-1412), and it is involved in many different hematological malignancies, including acute myeloid and lymphoblastic leukemia and myeloid dysplasia. syndrome) and is caused by several solid tumors (e.g., lung, breast, esophageal, gastric, colon, bile duct, pancreatic, ovarian, head and neck, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid cancer , endometrial cancer, neuroblastoma, rhabdomyosarcoma) (Haruo Sugiyama (2010) Jpn. J. Clin. Oncol. 40: 377-387). Vaccination against WT1 has resulted in objective anti-tumor responses in some cancer patients (Van Driessche et al. (2012) Oncologist 17: 250-259). Recently, clinical trials of isolated, expanded, and adoptively transferred WT1-specific T cells in patients with acute leukemia demonstrated safety and mediating antileukemic activity (Chapuis et al. (2013) Sci Transl Med 5: 174ra27).

However, the low frequency of high-affinity T cells that naturally respond to WT1 has so far limited the full utilization of this antigen in adoptive T cell therapy.

The identification of WT1-reactive T cells and the genetic sequences of WT1-specific TCRs has opened up several novel therapeutic opportunities.

The TCR gene sequence can be used in its native form, or by, for example, murineization of the constant TCR region or by cysteine modification of the human TCR constant region to promote correct pairing of TCR chains, or by optimization of the gene's code To modify its performance value.

Natural or modified TCR genes may be transferred in specific T cell subsets (including CD4 and/or CD8, naive, memory stem T cells, central memory cells, effector memory cells, or effector cells), or in other cell subsets , for example to promote different persistence lengths and different functions in engineered cells in vivo. TCR genes can also be transferred among T cell subsets with different polarizations (e.g., Th0/Tc0, Th1/Tc1, Th2/Tc2, Th17, Th22, or others), depending on what is best suited to target each possible tumor type the interleukin environment. In addition, these genes or chimeric genes designed to include the antigen-specific region of the TCR can be transferred in other cell subsets, including gamma/delta T cells, NK cells, NKT cells, hematopoietic stem cells, or other cells, to obtain therapeutic effect. It is further contemplated that natural or modified molecules designed to include the antigen-specific regions of a TCR may be engineered or coupled to non-cellular substrates (e.g., nanoparticles, exosomes, or other molecules), or may be used alone or with other molecules ( (e.g., toxins or antibodies) are used conjugated to soluble molecules, thereby exploiting their ability to recognize tumor cells, thereby conferring tumor specificity to the cytotoxic compound.

Gene transfer of novel TCRs, such as those described herein, into T lymphocytes is subject to certain limitations inherent to TCR biology. Specifically, tumor-specific alpha and beta TCR chains are expressed in lymphocytes that already harbor endogenous TCRs on the cell surface. Therefore, genetically modified cells exhibit competition for binding to at least two different TCRs of the CD3 complex, resulting in mutual TCR dilution and reduced T cell avidity and anti-tumor efficacy (Heemskerk, M.H. (2007) Blood 109: 235-243) . In addition, since TCR is a heterodimer, the α and β chains of the endogenous TCR may mismatch with the respective α and β chains of the transgenic TCR to create a new hybrid receptor with unpredictable and potentially harmful specificity (Bendle , G.M. (2010) Nature Medicine 16: 565-570; van Loenen, M.M (2010) Proceedings of the National Academy of Sciences of the United States of America 107: 10972-10977). These limitations, which represent major issues for TCR gene transfer-based adoptive immunotherapy in autologous and allogeneic settings, can be addressed by several strategies specifically aimed at improving TCR performance and promoting tumor-specific TCR Correct pairing between chains. These strategies include murineization of the constant region (Cohen C. J. (2006) Cancer Research 66: 8878-8886), cysteine modification of the constant region of tumor-specific TCR genes (Kuball J. (2007) Blood 109: 2331 -2338), or add siRNA designed to limit the expression of endogenous TCR genes (Okamoto S (2009) Cancer Research 69: 9003-9011). Our group demonstrated that endogenous TCR α and /or permanent disruption of the beta chain gene, allowing full expression of tumor-specific TCRs. This process, called TCR gene editing, proved superior to in vitro and in vivo TCR gene transfer (Provasi E., Genovese P. (2012) Nature Medicine 18: 807-15; Mastaglio S. et al. (2017) Blood 130: 606 -618). In addition, genome editing technology allows for the targeted integration of gene cassettes including tumor-specific TCR genes and promoter regions into endogenous genes destroyed by artificial nucleases (Lombardo A. (2007) Nature Biotechnology 25: 1298 -1306).

Finally, genome editing technology allows genetic disruption of multiple genes in a single cell: it is therefore conceivable that TCR gene editing could be combined with nuclease-based disruption of additional genes in target cells with the goal of improving the durability of WT1-specific cell products. properties, expansion, activity, resistance to exhaustion/senescence/inhibitory signals, homing ability or other functions. Therefore, based on a single antigen specificity, multiple therapeutic approaches can be envisioned, each tailored to the specific tumor type and tumor environment.

Example 2 Materials and Methods Isolation of WT1- specific T cells from patient samples was thawed at the institution in X-VIVO 15 (Euroclone/Lonza) supplemented with human serum 5%, penicillin/streptomycin 1%, and glutamine 1%. Bone marrow aspirate samples from 3 patients (Pt) diagnosed with acute myelogenous leukemia and who had undergone allogeneic hematopoietic transplantation were harvested and cryopreserved at the BioBank facility. A few hours after thawing, samples were washed in phosphate-buffered saline (Ca and Mg-free) supplemented with EDTA and 10% fetal calf serum, and subsequently incubated with dasatinib 50 nM in a total volume of 50 μl. Incubate for 30 minutes. After incubation, without washing, samples were stained with an HLA*0201-restricted APC-conjugated dextramer loaded with the VLDFAPPGA (SEQ ID NO: 72) (WT1) epitope and incubated on ice for 1.5 h.

Testing 2 different methods to isolate WT1-specific T cells: 1. 100 cells from patient 1 were directly sorted using a BD FACS Aria cell sorter in reverse transcription buffer (SmartScribe; Takara Clontech) in 1.5 ml Eppendorf tubes. Thereafter, the sample was heated at 65°C for 2 min, then heated on ice for 5 min and TCRαβ gene-specific cDNA synthesis was performed (Ruggiero E. et al. (2015) Nat. Commun. 6: 8081). 2. 500,000-2,000,000 cells from patients 1, 2, and 3 were stained with anti-APC magnetic beads (Miltenyi Biotec) according to the manufacturer's instructions, and positive selection was performed using the MACS system (Miltenyi Biotec). The positive fraction rich in WT1 VLDFAPPGA (SEQ ID NO: 72) specificity was then incubated in a solution supplemented with human serum 5%, penicillin/streptomycin 1%, glutamine 1%, IL-2 60 IU/ml, IL -7 Cultured in U-shaped bottom wells pre-coated with anti-CD3 and anti-CD28 monoclonal antibodies (1:2 ratio) in X-VIVO 15 (Euroclone/Lonza) at 5 ng/ml and IL-15 5 ng/ml . Medium was changed every 3-4 days and cells were divided if confluence was reached.

TCR profile sequencing was performed by extracting RNA from WT1-enriched T cells of patients 1, 2, and 3 using the Arcturus Pico Pure RNA extraction kit (Life Technology). Complementarity determining region (CDR) 3 sequences of WT1-specific T cells were amplified by using a modified RACE method (Ruggiero E. et al. (2015) Nat. Commun. 6: 8081). CDR3 homotypes were identified by sequencing the samples using an Illumina MiSeq sequencer and using MiXCR software (Bolotin, DA et al. (2015) Nature Methods 12: 380-381).

Results WT1-specific enrichment was detected in every individual patient analyzed. For patient 1, anti-VLDFAPPGA (SEQ ID NO: 72) (WT1) enrichment occurred at three different stimulation stages (2 weeks and 1 month after the first stimulation, and 1 month after the second stimulation). Detected by Dextramer staining by flow cytometry (Figure 13a). For patients 2 and 3, 2 growth communities specific for WT1 were detected, one from each patient, as assessed by flow cytometry with the APC-coupled Dextramer (Fig. 13b).

TCRαβ sequencing of WT1-specific T cells highlighted an increasing predominance of CDR3 homotypes defined by both TCR chains in each patient analyzed (Fig. 14a-d).

All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the above invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described means for carrying out the invention that are apparent to those skilled in cell biology, immunology, immunotherapy, molecular biology, oncology, or related fields are intended to be covered by the appended patent application. within the range.

Figure 1 Assessment of WT1-specific T cell expansion in 4 healthy donors over time. Four HD peripheral blood mononuclear cells were repeatedly stimulated with WT1 pool-137 (HD12, a) or WT1 HLA-A*02:01 pool (HD13-HD15, bd). Enrichment of antigen-reactive T cells was assessed by measuring IFNγ secretion and CD107a production in 6-hour co-cultures with autologous antigen-presenting cells (APCs) derived from WT1 protein pool pulse. In each test, unstimulated T cells, T cells in cocultures with APCs loaded with pools of unrelated peptides, and T cells treated with phorbol-12-myristate-13-acetate (PMA) were included. and ionomycin (not shown)-stimulated T cells served as controls. Dot plots indicate intracellular staining results for IFNγ production and CD107a exposure on the cell surface at a single time point (a, b, d) or over the entire culture time frame (c). HD, healthy donor; WT1, Wilms' tumor 1; PMA, phorbol 12-myristate 13-acetate; IFNγ, interferon-γ; S, stimulation. Figure 2 Identification of WT1 subpools and peptides recognized by expanded T lymphocytes isolated from each HD. Peptides inducing immune responses in T cells isolated from HD were evaluated using co-cultures of T cells with APCs loaded with 24 subpools (SP; HD12) or 11 peptides (HD13 and HD14). In addition, the experimental setup (not shown) included negative (unstimulated T cells and T cells co-cultured with APC loaded with a pool of non-related peptides or non-related peptides) and positive (cultured in the presence of PMA and ionomycin T cells) control. . IFNγ secretion and CD107a expression were assessed by cytofluorometric analysis. Representative dot plots are reported relative to co-cultures of T cells with subpool-loaded APCs and indicate the performance of IFNγ and CD107a. Dominant responses were observed in HD12 (a, b) for subpools 7 and 21, and in HD13 and HD14 (ce) for peptide 13. HD, healthy donor; SP, subpool; WT1, Wilms' tumor 1; APC, antigen-presenting cells; PMA, phorbol 12-myristate 13-acetate; IFNγ, interferon-γ. Figure 3 Epitope specificity of WT1-reactive T cells generated by sensitization with pooled peptides. To verify WT1 immunogenic peptides, T cells expanded from HD12 were co-cultured for 6 hours in the presence of APCs loaded with the peptide identified after deplotting on the mapping grid (a) and at least one unrelated peptide as a control. . Additionally, negative (unstimulated T cells) and positive (T cells cultured in the presence of PMA and ionomycin) controls were included in the experimental setup (not shown). Dot plots show the results of intracellular staining for IFNγ and surface CD107a for each HD. Enrichment of CD107a and IFNγ-positive cells was observed in T cells co-cultured with peptide 103, but not with unrelated peptides (b). WT1, Wilms' tumor 1; APC, antigen-presenting cells; PMA, 2; phorbol 12-myristate 13-acetate; IFNγ, interferon-γ. Figure 4 Computer prediction of HLA-peptide binding for different HDs. HLA typing results of HD12-HD14 (a). Report the results of computer prediction using NetMHC4.0 pan algorithm (b, HD12; c, HD13; d, HD14; e, HD15). Peptides identified as strong binders by the algorithm are highlighted in gray. HD, healthy donor; HLA, human leukocyte antigen. Figure 5 HLA restriction evaluation of identified WT1 immunogenic epitopes. To determine the HLA restriction elements of HD12, T cells were cocultured with different antigen-presenting EBV-BLCL cell lines, each carrying the specificity of interest shared with HD12 and pulsed with peptide 103 or an unrelated peptide as a control (a) HLA alleles. For HD13 and HD14, WT1-specific T cells were cocultured with T2 cells carrying the HLA-A*02:01 restriction element and pulsed with peptide 13 or an unrelated peptide as a control (b and c, respectively). The expression of CD107a labeling and IFNγ secretion were determined as readouts. HD, healthy donor; WT1, Wilm's tumor 1; IFNγ, interferon-γ; HLA, human leukocyte antigen; EBV, Epstein-Barr virus; BLCL, B lymphoblastoid cell Cell lines. Figure 6 Natural processing of immunogenic peptides. T cells isolated from HD13 (a) and HD14 (b) were compared with primary AML blasts from 3 different patients (indicated as pAML#15, pAML#15, pAML#16.1, pAML#16.2) co-culture. Co-cultures of blasts and irrelevant T cells were included as controls. Evaluate the percentage of caspase 3 (Cas3) expression in target (T) viable cells after 6 hours of co-culture with effector (E) T cells at different E:T ratios. Cas3 values obtained in control conditions were subtracted from Cas3 values obtained from co-cultures of primary blasts with HD13 and HD14 T cells. pAML, primary blast cells from patients with acute myeloid leukemia; HD, healthy donor; HLA, human leukocyte antigen; WT1, Wilm's tumor 1. Figure 7 WT1-specific TCR Vβ distribution characteristics. WT1-specific T cells isolated from HD were stained to quantify the TCR β-chain variable region (Vβ) profile by FACS analysis. The results indicate that highly dominant Vβ genes are expressed in HD12 and HD14, whereas for HD13, no clearly enriched and defined Vβ genes were detected. For HD15, Vβ immunodistribution analysis cannot be performed due to reduced cell fitness. TRBV, T cell receptor variable beta chain; HD, healthy donor; FACS, fluorescence-activated cell sorter. Figure 8 Selection tracking of WT1-specific TCR. TCRαβ sequencing of HD RNA was performed at different time points throughout the culture time frame. Sequencing results indicate the presence of major homologs of HD12 (a), HD13 (b), HD14 (c) and HD15 (d). The bar graph depicts the ten most significant CDR3 amino acid sequences identified at each time point (e.g., S4 corresponds to the sequencing results obtained after the fourth round of stimulation). For each bar, starting from the x-axis, the bottom segment represents the most important CDR3 sequence. The next nine most significant sequences are stacked above the bottom range and ordered by decreasing frequency of upward progression. The remaining sequences are grouped together at the top. CDR3, complementarity determining region 3; S, stimulation; HD, healthy donor; S, stimulation; HLA, human leukocyte antigen; P, peptide; RNA, ribonucleic acid. Figure 9 Functional affinity of TCR derived from HD12. After deletion of endogenous α and β chains, T cells from three different healthy donors were transduced with lentiviral vectors encoding TCR isolated from HD12. a) Evaluation of HD12-derived TCR performance by Vβ staining before and after Vβ enrichment. b) Functional affinity of HD12 source TCR. Effector cells were cocultured with EBV cell lines pulsed with NYESO-1 peptide as a control or reduced concentrations of peptide 103 (40 µg-0.4 pg, as indicated on the x-axis). After 6 hours of co-culture, the results showed the ability of HD12-edited T cells to recognize target cells in the presence of at least 0.4 µg of homologous peptide. No identification of unrelated peptides was measured. The expression of CD107a on CD8 T lymphocytes was assessed as a readout by flow cytometry. TCR, T cell receptor; HD, healthy donor; PBMC, peripheral blood mononuclear cells; NYESO-1, New York esophageal squamous cell carcinoma 1. Figure 10 Functional verification of HD13 source TCR. T cells from a healthy donor were transduced with a lentiviral vector encoding an HD13-derived TCR. a) Identification of WT1 pool by TCR isolated from HD13. Effector cells were co-cultured with T2 cell lines pulsed using either a WT1 pool or an unrelated pool as a control. In addition, negative (unstimulated T cells) and positive (T cells cultured in the presence of PMA and ionomycin) controls were included in the experimental setup. After 6 hours of co-culture, T cells were transduced with HD13-derived TCR to specifically recognize target cells pulsed with the WT1 pool by measuring IFNγ secretion on CD8 T cells. b) Test T cells in co-culture with T2 cells pulsed with WT1 source SP 1 and 14, both containing peptide 13 or SP 6 as negative control. The results show the ability of effector cells to specifically recognize SP1 and 14, as assessed by measuring IFNγ secretion and CD107a expression on CD8 T cells. HD, healthy donor; TCR, T cell receptor; WT1, Wilms' tumor 1; SP, subpool; PMA, 2; phorbol 12-myristate 13-acetate. Figure 11 Functional verification of TCR isolated from HD14. T cells isolated from a healthy donor were transduced with lentiviral vectors encoding HD14-derived TCR (TRAV12-2*01 WT and TRAV12-2*02 WT). a) Transduction efficiency of HD14 transferred T cells, expressed as Vβ expression on CD4 and CD8 T lymphocytes. b) Identification of WT1 pool by HD14 source TCR. Effector cells were co-cultured with T2 cell lines pulsed using either a WT1 pool or an unrelated pool as a control. After 6 hours of co-culture, results show the ability of HD14 transferred T cells to specifically recognize target cells pulsed with WT1 pools, as measured by assessment of IFNγ secretion on CD8 T cells. c) HD14-derived T cells recognize specific SP containing peptide 13. T cells were tested in co-cultures with T2 cells pulsed with WT13 sources SP 1 and 14, both containing peptide 13 or SP 6 as negative controls. Results demonstrate the ability of effector cells to specifically recognize SP1 and 14, as assessed by evaluation of CD107a expression and IFNγ secretion on CD8 T cells. HD, healthy donor; TCR, T cell receptor; WT1, Wilms' tumor 1; SP, subpool. Figure 12 HD14-derived TCR recognizes primary AML blasts. TCR-edited T cells from a healthy donor were transduced with lentiviral vectors encoding HD14-derived TCR (TRAV 12-2*02 WT and TRAV 12-2*02 mut). a) Evaluate the transduction efficiency of HD14 TCR by Vβ expression on CD4 and CD8 T cells. b) Compare edited T cells transduced with HD14 TCR TRAV 12-2*02 WT, TRAV12-2*02 mut or irrelevant TCR to patient-derived primary expressing high levels of WT1 and HLA-A02*01 restriction elements AML blast co-culture. In order to evaluate the survival rate of blast cells, the condition of target cells without T lymphocytes was included. Evaluate the percentage of caspase 3 (Cas3) expression in target (T) viable cells after 6 hours of co-culture with effector (E) T cells at different E:T ratios (as indicated in the figure). Cas3 values obtained from control conditions (T cells transduced with irrelevant TCR or blast cells alone) were subtracted from Cas3 values obtained from co-cultures of primary blasts with T cells harboring HD14-derived TCRs. pAML, primary blast cells from patients with acute myeloid leukemia; HD, healthy donor; HLA, human leukocyte antigen; WT1, Wilm's tumor 1. Figure 13 Identification of WT1-specific T cells by dextramer staining. Dot plots indicate different time points after T cell sorting (using an APC-labeled dextramer specific for the WT1 VLDFAPPGA peptide) and stimulation (patient 1, a) or at a single time point (b, patients 2 and 3) Dextramer staining results. WT1, Wilm's tumor 1. Figure 14 shows the TCR sequencing results of enriched WT1- specific T cells . T cells isolated from each patient and positively classified based on WT1 dextramer staining were characterized by TCRαβ sequencing. Sequencing results indicated the presence of major homologs for Patient 1 (a, b), Patient 2 (c) and Patient 3 (d). Bar graph depicts the ten most important CDR3 amino acid sequences identified for each patient and each TCR chain. For each bar, starting from the x-axis, the bottom segment represents the most important CDR sequence. The next nine most significant sequences are stacked above the bottom range and ordered by decreasing frequency of upward progression. The remaining sequences are grouped together at the top. WT1, Wilm's tumor 1; CDR3, complementarity determining region 3.

Claims (33)

A T cell receptor (TCR) that binds to Wilms tumor 1 protein (WT1) peptide when presented via the major histocompatibility complex (MHC), wherein the TCR: (i) Comprising CDR3α, which comprises the amino acid sequence of CASGGGADGLTF (SEQ ID NO: 25) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASGRGDTEAFF (SEQ ID NO: The amino acid sequence of 30) or its variant with up to three amino acid substitutions, additions or deletions; (ii) comprising CDR3α, which comprises the amino acid sequence of CAMRTGGGADGLTF (SEQ ID NO: 3) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSEAGLSYEQYF (SEQ ID NO: 8) The amino acid sequence or its variant with up to three amino acid substitutions, additions or deletions; (iii) Comprising CDR3α, which comprises the amino acid sequence of CILSTRVWAGSYQLTF (SEQ ID NO: 14) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CATGQATQETQYF (SEQ ID NO: The amino acid sequence of 19) or its variant with up to three amino acid substitutions, additions or deletions; (iv) comprising CDR3α, which comprises the amino acid sequence of CAVIGGTDSWGKLQF (SEQ ID NO: 36) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSQEEGAVYGYTF (SEQ ID NO: The amino acid sequence of 41) or its variant with up to three amino acid substitutions, additions or deletions; (v) comprising CDR3α, which comprises the amino acid sequence of CAVIGGTDSWGKLQF (SEQ ID NO: 36) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CATSREGLAADTQYF (SEQ ID NO: The amino acid sequence of 52) or its variant with up to three amino acid substitutions, additions or deletions; (vi) Comprising CDR3α, which comprises the amino acid sequence of CVVPRGLSTDSWGKLQF (SEQ ID NO: 47) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CATSREGLAADTQYF (SEQ ID NO: The amino acid sequence of 52) or its variant with up to three amino acid substitutions, additions or deletions; (vii) comprising CDR3α, which comprises the amino acid sequence of CVVPRGLSTDSWGKLQF (SEQ ID NO: 47) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSQEEGAVYGYTF (SEQ ID NO: The amino acid sequence of 41) or its variant with up to three amino acid substitutions, additions or deletions; (viii) comprising CDR3α, which comprises the amino acid sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSSGLAFYEQYF (SEQ ID NO: The amino acid sequence of 98) or its variants with up to three amino acid substitutions, additions or deletions; (ix) comprising CDR3α, which comprises the amino acid sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSQLSGRDSYEQYF (SEQ ID NO: The amino acid sequence of 104) or its variant with up to three amino acid substitutions, additions or deletions; (x) comprising CDR3α, which comprises the amino acid sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSTLGGELFF (SEQ ID NO: The amino acid sequence of 120) or its variant with up to three amino acid substitutions, additions or deletions; (xi) comprising CDR3α, which comprises the amino acid sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSTLGGELFF (SEQ ID NO: The amino acid sequence of 120) or its variant with up to three amino acid substitutions, additions or deletions; (xii) comprising CDR3α, which comprises the amino acid sequence of CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSLEGRAMPRDSHQETQYF (SEQ ID NO: The amino acid sequence of 136) or its variants with up to three amino acid substitutions, additions or deletions; (xiii) comprising CDR3α, which comprises the amino acid sequence of CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CATSWGLNEQYF (SEQ ID NO: The amino acid sequence of 142) or its variants with up to three amino acid substitutions, additions or deletions; (xiv) comprising CDR3α, which comprises the amino acid sequence of CAATSRDDMRF (SEQ ID NO: 131) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSLEGRAMPRDSHQETQYF (SEQ ID NO: The amino acid sequence of 136) or its variants with up to three amino acid substitutions, additions or deletions; (xv) comprises CDR3α, which comprises the amino acid sequence of CAATSRDDMRF (SEQ ID NO: 131) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CATSWGLNEQYF (SEQ ID NO: The amino acid sequence of 142) or its variants with up to three amino acid substitutions, additions or deletions; (xvi) Comprises CDR3α, which comprises the amino acid sequence of CALPDKVIF (SEQ ID NO: 148) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSVSAGSTGELFF (SEQ ID NO: The amino acid sequence of 158) or its variant with up to three amino acid substitutions, additions or deletions; (xvii) comprising CDR3α, which comprises the amino acid sequence of CAGLYATNKLIF (SEQ ID NO: 153) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSVSAGSTGELFF (SEQ ID NO: The amino acid sequence of 158) or its variant with up to three amino acid substitutions, additions or deletions; (xviii) comprising CDR3α, which comprises the amino acid sequence of CAAPNDYKLSF (SEQ ID NO: 93) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSTLGGELFF (SEQ ID NO: The amino acid sequence of 120) or its variant with up to three amino acid substitutions, additions or deletions; (xix) comprising CDR3α, which comprises the amino acid sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSSGLAFYEQYF (SEQ ID NO: The amino acid sequence of 98) or its variants with up to three amino acid substitutions, additions or deletions; (xx) comprises CDR3α, which comprises the amino acid sequence of CAVRDGGATNKLIF (SEQ ID NO: 110) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSQLSGRDSYEQYF (SEQ ID NO: The amino acid sequence of 104) or its variant with up to three amino acid substitutions, additions or deletions; (xxi) Comprises CDR3α, which comprises the amino acid sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSSGLAFYEQYF (SEQ ID NO: 98) or a variant thereof having up to three amino acid substitutions, additions or deletions; or (xxii) comprising CDR3α, which comprises the amino acid sequence of CLVGGYTGGFKTIF (SEQ ID NO: 115) or a variant thereof with up to three amino acid substitutions, additions or deletions; and CDR3β, which comprises CASSQLSGRDSYEQYF (SEQ ID NO: The amino acid sequence of 104) or its variant with up to three amino acid substitutions, additions or deletions. For example, the TCR of request item 1 contains the following CDR sequence: (i) CDR1α-NSAFQY (SEQ ID NO: 23), CDR2α-TYSSGN (SEQ ID NO: 24), CDR3α - CASGGGADGLTF (SEQ ID NO: 25), CDR1β-SGDLS (SEQ ID NO: 28), CDR2β-YYNGEE (SEQ ID NO: 29), and CDR3β-CASGRGDTEAFF (SEQ ID NO: 30), or variants thereof each having up to three amino acid substitutions, additions or deletions; (ii)CDR1α-TSDQSYG (SEQ ID NO: 1), CDR2α - QGSYDEQN (SEQ ID NO: 2), CDR3α - CAMRTGGGADGLTF (SEQ ID NO: 3), CDR1β-SNHLY (SEQ ID NO: 6), CDR2β - FYNNEI (SEQ ID NO: 7), and CDR3β - CASSEAGLSYEQYF (SEQ ID NO: 8), or variants thereof each having up to three amino acid substitutions, additions or deletions; (iii)CDR1α-TISGTDY (SEQ ID NO: 12), CDR2α-GLTSN (SEQ ID NO: 13), CDR3α - CILSTRVWAGSYQLTF (SEQ ID NO: 14), CDR1β-KGHDR (SEQ ID NO: 17), CDR2β-SFDVKD (SEQ ID NO: 18), and CDR3β-CATGQATQETQYF (SEQ ID NO: 19), or variants thereof each having up to three amino acid substitutions, additions or deletions; (iv) CDR1α-DRGSQS (SEQ ID NO: 34), CDR2α-IYSNGD (SEQ ID NO: 35), CDR3α - CAVIGGTDSWGKLQF (SEQ ID NO: 36), CDR1β - LGHNA (SEQ ID NO: 39), CDR2β-YSLEER (SEQ ID NO: 40), and CDR3β - CASSQEEGAVYGYTF (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions or deletions; (v)CDR1α-DRGSQS (SEQ ID NO: 34), CDR2α-IYSNGD (SEQ ID NO: 35), CDR3α - CAVIGGTDSWGKLQF (SEQ ID NO: 36), CDR1β-LNHNV (SEQ ID NO: 50), CDR2β - YYDKDF (SEQ ID NO: 51), and CDR3β - CATSREGLAADTQYF (SEQ ID NO: 52), or variants thereof each having up to three amino acid substitutions, additions or deletions; (vi)CDR1α-NSASQS (SEQ ID NO: 45), CDR2α - VYSSGN (SEQ ID NO: 46), CDR3α - CVVPRGLSTDSWGKLQF (SEQ ID NO: 47), CDR1β-LNHNV (SEQ ID NO: 50), CDR2β - YYDKDF (SEQ ID NO: 51), and CDR3β - CATSREGLAADTQYF (SEQ ID NO: 52), or variants thereof each having up to three amino acid substitutions, additions or deletions; (vii) CDR1α-NSASQS (SEQ ID NO: 45), CDR2α - VYSSGN (SEQ ID NO: 46), CDR3α - CVVPRGLSTDSWGKLQF (SEQ ID NO: 47), CDR1β - LGHNA (SEQ ID NO: 39), CDR2β-YSLEER (SEQ ID NO: 40), and CDR3β - CASSQEEGAVYGYTF (SEQ ID NO: 41), or variants thereof each having up to three amino acid substitutions, additions or deletions; (viii)CDR1α-VSNAYN (SEQ ID NO: 91), CDR2α-GSKP (SEQ ID NO: 92), CDR3α-CAAPNDYKLSF (SEQ ID NO: 93), CDR1β-SEHNR (SEQ ID NO: 96), CDR2β - FQNEAQ (SEQ ID NO: 97), and CDR3β - CASSSGLAFYEQYF (SEQ ID NO: 98), or variants thereof each having up to three amino acid substitutions, additions or deletions; (ix)CDR1α-VSNAYN (SEQ ID NO: 91), CDR2α-GSKP (SEQ ID NO: 92), CDR3α-CAAPNDYKLSF (SEQ ID NO: 93), CDR1β-SGHDN (SEQ ID NO: 102), CDR2β-FVKESK (SEQ ID NO: 103), and CDR3β - CASSQLSGRDSYEQYF (SEQ ID NO: 104), or variants thereof each having up to three amino acid substitutions, additions or deletions; (x)CDR1α-VSGNPY (SEQ ID NO: 108), CDR2α - YITGDNLV (SEQ ID NO: 109), CDR3α - CAVRDGGATNKLIF (SEQ ID NO: 110), CDR1β-MNHEY (SEQ ID NO: 118), CDR2β-SMNVEV (SEQ ID NO: 119), and CDR3β-CASSTLGGELFF (SEQ ID NO: 120), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xi)CDR1α-NIATNDY (SEQ ID NO: 113), CDR2α - GYKTK (SEQ ID NO: 114), CDR3α - CLVGGYTGGFKTIF (SEQ ID NO: 115), CDR1β-MNHEY (SEQ ID NO: 118), CDR2β-SMNVEV (SEQ ID NO: 119), and CDR3β-CASSTLGGELFF (SEQ ID NO: 120), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xii) CDR1α-SSVSVY (SEQ ID NO: 124), CDR2α-YLSGSTLV (SEQ ID NO: 125), CDR3α - CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126), CDR1β-SEHNR (SEQ ID NO: 134), CDR2β - FQNEAQ (SEQ ID NO: 135), and CDR3β - CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xiii)CDR1α-SSVSVY (SEQ ID NO: 124), CDR2α-YLSGSTLV (SEQ ID NO: 125), CDR3α - CAVTLLSIEPSAGGYQKVTF (SEQ ID NO: 126), CDR1β-LNHNV (SEQ ID NO: 140), CDR2β - YYDKDF (SEQ ID NO: 141), and CDR3β-CATSWGLNEQYF (SEQ ID NO: 142), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xiv)CDR1α-DSASNY (SEQ ID NO: 129), CDR2α - IRSNVGE (SEQ ID NO: 130), CDR3α-CAATSRDDMRF (SEQ ID NO: 131), CDR1β-SEHNR (SEQ ID NO: 134), CDR2β - FQNEAQ (SEQ ID NO: 135), and CDR3β - CASSLEGRAMPRDSHQETQYF (SEQ ID NO: 136), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xv)CDR1α-DSASNY (SEQ ID NO: 129), CDR2α - IRSNVGE (SEQ ID NO: 130), CDR3α-CAATSRDDMRF (SEQ ID NO: 131), CDR1β-LNHNV (SEQ ID NO: 140), CDR2β - YYDKDF (SEQ ID NO: 141), and CDR3β-CATSWGLNEQYF (SEQ ID NO: 142), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xvi)CDR1α-TRDTTYY (SEQ ID NO: 146), CDR2α-RNSFDEQN (SEQ ID NO: 147), CDR3α - CALPDKVIF (SEQ ID NO: 148), CDR1β-SGDLS (SEQ ID NO: 156), CDR2β-YYNGEE (SEQ ID NO: 157), and CDR3β - CASSSVAGSTGELFF (SEQ ID NO: 158), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xvii)CDR1α-SIFNT (SEQ ID NO: 151), CDR2α - LYKAGEL (SEQ ID NO: 152), CDR3α - CAGLYATNKLIF (SEQ ID NO: 153), CDR1β-SGDLS (SEQ ID NO: 156), CDR2β-YYNGEE (SEQ ID NO: 157), and CDR3β - CASSSVAGSTGELFF (SEQ ID NO: 158), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xviii)CDR1α-VSNAYN (SEQ ID NO:91), CDR2α-GSKP (SEQ ID NO: 92), CDR3α-CAAPNDYKLSF (SEQ ID NO: 93), CDR1β-MNHEY (SEQ ID NO: 118), CDR2β-SMNVEV (SEQ ID NO: 119), and CDR3β-CASSTLGGELFF (SEQ ID NO: 120), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xix)CDR1α-VSGNPY (SEQ ID NO: 108), CDR2α - YITGDNLV (SEQ ID NO: 109), CDR3α - CAVRDGGATNKLIF (SEQ ID NO: 110), CDR1β-SEHNR (SEQ ID NO: 96), CDR2β - FQNEAQ (SEQ ID NO: 97), and CDR3β - CASSSGLAFYEQYF (SEQ ID NO: 98), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xx)CDR1α-VSGNPY (SEQ ID NO: 108), CDR2α - YITGDNLV (SEQ ID NO: 109), CDR3α - CAVRDGGATNKLIF (SEQ ID NO: 110), CDR1β-SGHDN (SEQ ID NO: 102), CDR2β-FVKESK (SEQ ID NO: 103), and CDR3β - CASSQLSGRDSYEQYF (SEQ ID NO: 104), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xxi)CDR1α-NIATNDY (SEQ ID NO: 113), CDR2α - GYKTK (SEQ ID NO: 114), CDR3α - CLVGGYTGGFKTIF (SEQ ID NO: 115), CDR1β-SEHNR (SEQ ID NO: 96), CDR2β - FQNEAQ (SEQ ID NO: 97), and CDR3β - CASSSGLAFYEQYF (SEQ ID NO: 98), or variants thereof each having up to three amino acid substitutions, additions or deletions; (xxii)CDR1α-NIATNDY (SEQ ID NO: 113), CDR2α - GYKTK (SEQ ID NO: 114), CDR3α - CLVGGYTGGFKTIF (SEQ ID NO: 115), CDR1β-SGHDN (SEQ ID NO: 102), CDR2β-FVKESK (SEQ ID NO: 103), and CDR3β - CASSQLSGRDSYEQYF (SEQ ID NO: 104), or variants thereof each having up to three amino acid substitutions, additions or deletions; or (xxiii)CDR1α-DRGSQS (SEQ ID NO: 182), CDR2α-IYSNGD (SEQ ID NO: 183), CDR3α - CASGGGADGLTF (SEQ ID NO: 25), CDR1β-SGDLS (SEQ ID NO: 28), CDR2β-YYNGEE (SEQ ID NO: 29), and CDR3β-CASGRGDTEAFF (SEQ ID NO: 30), or variants thereof each having up to three amino acid substitutions, additions or deletions. For example, the TCR of request item 1 or 2 includes: (i) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising SEQ ID NO: The amino acid sequence of 31 or its variant with at least 75% sequence identity; (ii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof having at least 75% sequence identity; and a beta chain variable domain comprising SEQ ID NO: The amino acid sequence of 9 or its variant with at least 75% sequence identity; (iii) an alpha chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 15 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain, which comprises SEQ ID NO: 20 amino acid sequences or variants thereof that have at least 75% sequence identity; (iv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising SEQ ID NO: The amino acid sequence of 42 or its variant with at least 75% sequence identity; (v) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 37 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising SEQ ID NO: The amino acid sequence of 53 or its variant with at least 75% sequence identity; (vi) an alpha chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 48 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain, which comprises SEQ ID NO: The amino acid sequence of 53 or its variant with at least 75% sequence identity; (vii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 48 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising SEQ ID NO: The amino acid sequence of 42 or its variant with at least 75% sequence identity; (viii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 94 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising SEQ ID NO: 99 amino acid sequence or a variant thereof that has at least 75% sequence identity; (ix) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 94 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising SEQ ID NO: The amino acid sequence of 105 or its variant with at least 75% sequence identity; (x) an alpha chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 111 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain, which comprises SEQ ID NO: The amino acid sequence of 121 or its variant with at least 75% sequence identity; (xi) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 116 or a variant thereof having at least 75% sequence identity; and a beta chain variable domain comprising SEQ ID NO: The amino acid sequence of 121 or its variant with at least 75% sequence identity; (xii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 127 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising SEQ ID NO: The amino acid sequence of 137 or its variant with at least 75% sequence identity; (xiii) an alpha chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 127 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain, which comprises SEQ ID NO: The amino acid sequence of 143 or its variant with at least 75% sequence identity; (xiv) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 132 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising SEQ ID NO: The amino acid sequence of 137 or its variant with at least 75% sequence identity; (xv) an α-chain variable domain comprising the amino acid sequence of SEQ ID NO: 132 or a variant thereof having at least 75% sequence identity thereto; and a β-chain variable domain comprising SEQ ID NO: The amino acid sequence of 143 or its variant with at least 75% sequence identity; (xvi) an alpha chain variable domain, comprising the amino acid sequence of SEQ ID NO: 149 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain, comprising SEQ ID NO: The amino acid sequence of 159 or its variant with at least 75% sequence identity; (xvii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising SEQ ID NO: The amino acid sequence of 159 or its variant with at least 75% sequence identity; (xviii) α chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 94 or a variant thereof having at least 75% sequence identity thereto; and β chain variable domain, which comprises SEQ ID NO: The amino acid sequence of 121 or its variant with at least 75% sequence identity; (xix) an α-chain variable domain comprising the amino acid sequence of SEQ ID NO: 111 or a variant thereof having at least 75% sequence identity; and a β-chain variable domain comprising SEQ ID NO: 99 amino acid sequence or a variant thereof that has at least 75% sequence identity; (xx) α chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 111 or a variant thereof having at least 75% sequence identity thereto; and β chain variable domain, which comprises SEQ ID NO: The amino acid sequence of 105 or its variant with at least 75% sequence identity; (xxi) α chain variable domain, which comprises the amino acid sequence of SEQ ID NO: 116 or a variant thereof having at least 75% sequence identity thereto; and β chain variable domain, which comprises SEQ ID NO: 99 amino acid sequence or a variant thereof that has at least 75% sequence identity; (xxii) an alpha chain variable domain comprising the amino acid sequence of SEQ ID NO: 116 or a variant thereof having at least 75% sequence identity thereto; and a beta chain variable domain comprising SEQ ID NO: The amino acid sequence of 105 or its variant with at least 75% sequence identity; (xxiii) α-chain variable domain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 185, 190 or a variant thereof having at least 75% sequence identity; and β-chain variable domain , which comprises the amino acid sequence of SEQ ID NO: 31 or a variant thereof having at least 75% sequence identity thereto. For example, the TCR of any of the aforementioned requests includes: (i) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 27 or a variant thereof with at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 32, SEQ ID NO: 33. The amino acid sequence of the group consisting of SEQ ID NO: 203 and variants of SEQ ID NO: 32, 33 and 203 that have at least 75% sequence identity therewith; (ii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 5 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 11. The amino acid sequence of the group consisting of SEQ ID NO: 195 and variants of SEQ ID NO: 10, 11 and 195 that have at least 75% sequence identity therewith; (iii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 16 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 21, SEQ ID NO: 22. The amino acid sequence of the group consisting of SEQ ID NO: 197 and the variants of SEQ ID NO: 21, 22 and 197 that have at least 75% sequence identity therewith; (iv) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 38 or a variant thereof with at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 43, SEQ ID NO: 44. The amino acid sequence of the group consisting of SEQ ID NO: 215 and variants of SEQ ID NO: 43, 44 and 215 having at least 75% sequence identity therewith; (v) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 38 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 54, SEQ ID NO: 55. The amino acid sequence of the group consisting of SEQ ID NO: 217 and variants of SEQ ID NO: 54, 55 and 217 having at least 75% sequence identity therewith; (vi) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 49 or a variant thereof with at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 54, SEQ ID NO: 55. The amino acid sequence of the group consisting of SEQ ID NO: 217 and variants of SEQ ID NO: 54, 55 and 217 having at least 75% sequence identity therewith; (vii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 49 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 43, SEQ ID NO: 44. The amino acid sequence of the group consisting of SEQ ID NO: 215 and variants of SEQ ID NO: 43, 44 and 215 having at least 75% sequence identity therewith; (viii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 95 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 100, SEQ ID NO: Amino acid sequences of the group consisting of 101 and variants of SEQ ID NO: 100 and 101 having at least 75% sequence identity therewith; (ix) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 95 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 106, SEQ ID NO: Amino acid sequences of the group consisting of 107 and variants of SEQ ID NO: 106 and 107 having at least 75% sequence identity therewith; (x) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 112 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 122, SEQ ID NO: Amino acid sequences of the group consisting of 123 and variants of SEQ ID NO: 122 and 123 having at least 75% sequence identity therewith; (xi) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 117 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 122, SEQ ID NO: Amino acid sequences of the group consisting of 123 and variants of SEQ ID NO: 122 and 123 having at least 75% sequence identity therewith; (xii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 128 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 138, SEQ ID NO: Amino acid sequences of the group consisting of 139 and variants of SEQ ID NO: 138 and 139 having at least 75% sequence identity therewith; (xiii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 128 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 144, SEQ ID NO: Amino acid sequences of the group consisting of 145 and variants of SEQ ID NO: 144 and 145 having at least 75% sequence identity therewith; (xiv) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 133 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 138, SEQ ID NO: Amino acid sequences of the group consisting of 139 and variants of SEQ ID NO: 138 and 139 having at least 75% sequence identity therewith; (xv) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 133 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 144, SEQ ID NO: Amino acid sequences of the group consisting of 145 and variants of SEQ ID NO: 144 and 145 having at least 75% sequence identity therewith; (xvi) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 150 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 160, SEQ ID NO: Amino acid sequences of the group consisting of 161 and variants of SEQ ID NO: 160 and 161 having at least 75% sequence identity therewith; (xvii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 155 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 160, SEQ ID NO: Amino acid sequences of the group consisting of 161 and variants of SEQ ID NO: 160 and 161 having at least 75% sequence identity therewith; (xviii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 95 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 122, SEQ ID NO: Amino acid sequences of the group consisting of 123 and variants of SEQ ID NO: 122 and 123 having at least 75% sequence identity therewith; (xix) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 112 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 100, SEQ ID NO: Amino acid sequences of the group consisting of 101 and variants of SEQ ID NO: 100 and 101 having at least 75% sequence identity therewith; (xx) α chain, which includes the amino acid sequence of SEQ ID NO: 112 or a variant thereof having at least 75% sequence identity; and β chain, which includes the amino acid sequence selected from SEQ ID NO: 106, SEQ ID NO: Amino acid sequences of the group consisting of 107 and variants of SEQ ID NO: 106 and 107 having at least 75% sequence identity therewith; (xxi) α chain, which includes the amino acid sequence of SEQ ID NO: 117 or a variant thereof with at least 75% sequence identity; and β chain, which includes the amino acid sequence selected from SEQ ID NO: 100, SEQ ID NO: Amino acid sequences of the group consisting of 101 and variants of SEQ ID NO: 100 and 101 having at least 75% sequence identity therewith; (xxii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 117 or a variant thereof with at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 106, SEQ ID NO: Amino acid sequences of the group consisting of 107 and variants of SEQ ID NO: 106 and 107 having at least 75% sequence identity therewith; (xxiii) (a) α chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 186, 191, 198, 199, 200, 201, 202 and a SEQ ID with at least 75% sequence identity thereto Variants of NO: 186, 191, 198, 199, 200, 201 and 202; and a beta chain comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least 75% sequence identity; (b) α chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 186, 191, 198, 199, 200, 201, 202 and SEQ ID NO: 186 having at least 75% sequence identity therewith , 191, 198, 199, 200, 201 and 202 variants; and a beta chain comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof having at least 75% sequence identity thereto; or (c) Alpha chain, which includes an amino acid sequence selected from the group consisting of SEQ ID NO: 186, 191, 198, 199, 200, 201, 202 and SEQ ID NO: 186 having at least 75% sequence identity therewith. , 191, 198, 199, 200, 201 and 202 variants; and a β chain, which includes the amino acid sequence of SEQ ID NO: 203 or a variant thereof that has at least 75% sequence identity; (xxiv) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 194 or a variant thereof with at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 11. The amino acid sequence of the group consisting of SEQ ID NO: 195 and variants of SEQ ID NO: 10, 11 and 195 that have at least 75% sequence identity therewith; (xxv) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 196 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 21, SEQ ID NO: 22. The amino acid sequence of the group consisting of SEQ ID NO: 197 and the variants of SEQ ID NO: 21, 22 and 197 that have at least 75% sequence identity therewith; (xxvi) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 214 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 43, SEQ ID NO: 44. The amino acid sequence of the group consisting of SEQ ID NO: 215 and variants of SEQ ID NO: 43, 44 and 215 having at least 75% sequence identity therewith; (xxvii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 214 or a variant thereof with at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 54, SEQ ID NO: 55. The amino acid sequence of the group consisting of SEQ ID NO: 217 and variants of SEQ ID NO: 54, 55 and 217 having at least 75% sequence identity therewith; (xxviii) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 216 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 54, SEQ ID NO: 55. The amino acid sequence of the group consisting of SEQ ID NO: 217 and variants of SEQ ID NO: 54, 55 and 217 having at least 75% sequence identity therewith; or (xxix) an alpha chain, which includes the amino acid sequence of SEQ ID NO: 216 or a variant thereof having at least 75% sequence identity; and a beta chain, which includes an amino acid sequence selected from SEQ ID NO: 43, SEQ ID NO: 44. The amino acid sequence of the group consisting of SEQ ID NO: 215 and variants of SEQ ID NO: 43, 44 and 215 having at least 75% sequence identity therewith. A T cell receptor (TCR) that binds to a Wilms' tumor 1 protein (WT1) peptide when presented by a major histocompatibility complex (MHC), wherein the WT1 peptide comprises a protein selected from the group consisting of GAQYRIHTHGVFRGI (SEQ ID NO. : 181), LLAAILDFLLLQDPA (SEQ ID NO: 82) and CMTWNQMNLGATLKG (SEQ ID NO: 87), and their respective variants with up to three amino acid substitutions, additions or deletions. The TCR of any one of the preceding claims, which binds to MHC I and/or MHC II peptide complexes. A TCR as in any one of the preceding claims, which is limited to human leukocyte antigen (HLA) alleles, preferably wherein the TCR is limited to HLA-A, HLA-B or HLA-C alleles, more preferably where the TCR is limited to HLA-A*02:01, HLA-B*38:01, HLA-C*03:03 or HLA-C*07:02. A TCR based on the following: a. Part (ii) or (xxiv) of any one of claims 1 to 4, which is limited to HLA-B*38:01 or HLA-C*07:02; b. Parts (iii), (viii) to (xxii) or (xxv) of any of claims 1 to 4, which are limited to HLA-A*02:01; or c. Part (i) or (xxiii) of any one of claims 1 to 4, which is limited to HLA-A*02:01 or HLA-C*03:03. The TCR of any one of the preceding claims, which includes one or more mutations at the α chain/β chain interface, such that when the α chain and the β chain are expressed in T cells, these chains are not identical to the endogenous TCR The frequency of mismatches between α and β chains is reduced. The TCR of claim 9, wherein the one or more mutations introduce cysteine residues into the constant region domain of each of the alpha chain and the beta chain, wherein the cysteine residues A disulfide bond can be formed between the alpha chain and the beta chain. The TCR of any one of the preceding claims, which includes a murine constant region. The TCR of any one of the preceding claims, wherein the TCR is a soluble TCR. An isolated polynucleotide encoding the alpha chain of a T cell receptor (TCR) according to any one of the preceding claims and/or the beta chain of a TCR according to any one of the preceding claims. The isolated polynucleotide of claim 13, wherein the polynucleotide encodes the alpha chain linked to the beta chain. For example, the isolated polynucleotide of claim 13 or 14 further encodes one or more short interfering RNA (siRNA) or other agents capable of reducing or preventing the expression of one or more endogenous TCR genes. A vector comprising the polynucleotide of any one of claims 13 to 15. The vector of claim 16, which includes polynucleotides encoding one or more CD3 chains, CD8, suicide genes and/or selectable markers. A cell comprising a TCR as in any one of claims 1 to 11, a polynucleotide as in any one of claims 13 to 15, or a vector as in claim 16 or 17, Optionally, the cell further comprises a vector encoding one or more CD3 chains, CD8, suicide genes and/or selectable markers. For example, the cell of claim 18, wherein the cell is a T cell, a lymphocyte or a stem cell, optionally the T cell, the lymphocyte or the stem cell is selected from the group consisting of: CD4 cells, CD8 cells, naive T cells, Memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, γ/ δ T cells, natural killer (NK) cells, natural killer T (NKT) cells, cytokine-induced killer (CIK) cells, hematopoietic stem cells and pluripotent stem cells. The cell of claim 19, wherein the cell is a T cell that has been isolated from an individual. The cell of any one of claims 18 to 20, wherein the endogenous gene encoding the TCR alpha chain and/or the endogenous gene encoding the TCR beta chain is disrupted, preferably such that the endogenous gene encoding the TCR alpha chain and /or the endogenous gene encoding the TCR β chain is not expressed, optionally in which the expression cassette encoding the TCR α chain is destroyed by inserting an expression cassette containing a polynucleotide sequence encoding the TCR of any one of claims 1 to 11 The endogenous gene and/or the endogenous gene encoding the TCR β chain, further optionally destroys one or more endogenous genes encoding MHC, and further optionally participates in persistence, amplification, activity, failure/aging/ Endogenous genes for resistance to inhibitory signals, homing ability or other T cell functions are disrupted, preferably those involved in persistence, expansion, activity, resistance to exhaustion/senescence/inhibitory signals, homing Endogenous genes for nesting ability or other T cell functions are selected from the group consisting of: PD1 , TIM3 , LAG3 , 2B4 , KLRG1 , TGFbR , CD160 , TIGIT , CTLA4 and CD39 . A method of preparing cells, which includes the step of introducing the vector of claims 16 and/or 17 into cells in vitro, ex vivo or in vivo, for example by transfection or transduction. The method of claim 22, which includes a step of T cell editing, which step includes using an artificial nuclease to destroy the endogenous gene encoding the TCR α chain and/or the endogenous gene encoding the TCR β chain, preferably wherein the artificial nucleic acid The enzyme system is selected from the group consisting of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas systems. The method of claim 23, which includes the step of targeting the expression cassette into the endogenous gene encoding the TCR α chain and/or the endogenous gene encoding the TCR β chain destroyed by the artificial nuclease, wherein the The expression cassette contains a polynucleotide sequence encoding a TCR according to any one of claims 1 to 11. The method of any one of claims 22 to 24, which includes the step of destroying one or more endogenous genes encoding MHC, preferably wherein the cell line prepared by the method does not cause allogeneic reactions. T cells. The method of any one of claims 22 to 25, comprising disrupting one or more endogenous genes to modify persistence, amplification, activity, resistance to exhaustion/senescence/inhibitory signals, homing ability, or Steps of other T cell functions, preferably wherein the method includes integrating expression cassettes into cells involved in persistence, expansion, activity, resistance to exhaustion/senescence/inhibitory signals, homing ability or other T cell functions and The step in the endogenous gene destroyed by artificial nuclease, wherein the expression cassette contains a polynucleotide sequence encoding the TCR of any one of claims 1 to 11, preferably wherein the endogenous gene is selected from the following composition Groups: PD1 , TIM3 , LAG3 , 2B4 , KLRG1 , TGFbR , CD160 , TIGIT , CTLA4 and CD39 . For example, the cells of any one of claims 18 to 21 or the cells prepared by the method of any one of claims 22 to 26 are used for adoptive cell transfer, preferably adoptive T cell transfer, depending on In this case, the adoptive T cell transfer is an allogeneic adoptive T cell transfer, an autologous adoptive T cell transfer, or a universal adoptive T cell transfer that does not cause an allogeneic reaction. A chimeric molecule comprising a TCR or part thereof as in any one of claims 1 to 11, coupled to a non-cellular substrate, a toxin and/or an antibody, optionally wherein the non-cellular substrate is selected from the following Groups: Nanoparticles, exosomes and other non-cellular substrates. The TCR of any one of claims 1 to 11, the isolated polynucleotide of any one of claims 13 to 15, the vector of claim 16 or 17, the vector of any one of claims 18 to 21 Cells, cells prepared by the method of any one of claims 22 to 26, or chimeric molecules of claim 28 for use in therapy. The TCR of any one of claims 1 to 11, the isolated polynucleotide of any one of claims 13 to 15, the vector of claim 16 or 17, the vector of any one of claims 18 to 21 Cells, cells prepared by the method of any one of claims 22 to 26, or chimeric molecules of claim 28, for the treatment and/or prevention of diseases associated with the expression of WT1, as appropriate, wherein The disease associated with expression of WT1 is a proliferative disorder, preferably wherein the proliferative disorder is a hematological malignancy or a solid tumor, preferably wherein the hematological malignancy is selected from the group consisting of: acute myeloid leukemia (AML) , chronic myelogenous leukemia (CML), lymphoblastic leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin's lymphoma; or older Preferably, the solid tumor is selected from the group consisting of: lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, cholangiocarcinoma, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, thyroid Carcinoma, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial cancer, biliary tract cancer, glioblastoma, mesothelioma, cervix cancer and colorectal cancer. A method of treating and/or preventing diseases related to the manifestations of WT1, which includes administering a TCR as in any one of claims 1 to 11, or a treatment as in any one of claims 13 to 15 to an individual in need thereof An isolated polynucleotide, a vector as claimed in claim 16 or 17, a cell as claimed in any one of claims 18 to 21, a cell prepared by a method as claimed in any one of claims 22 to 26, or as claimed in claim 16 Step 28 for chimeric molecules. The method of claim 31, wherein the disease associated with the expression of WT1 is a proliferative disorder, preferably wherein the proliferative disorder is a hematological malignancy or a solid tumor, preferably wherein the hematological malignancy is selected from the group consisting of : Acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), lymphoblastic leukemia, myelodysplastic syndrome, lymphoma, multiple myeloma, non-Hodgkin's lymphoma and Hodgkin's lymphoma ; Or preferably, the solid tumor is selected from the group consisting of: lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, bile duct cancer, pancreatic cancer, ovarian cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma thyroid cancer, endometrial cancer, neuroblastoma, rhabdomyosarcoma, liver cancer, melanoma, prostate cancer, kidney cancer, soft tissue sarcoma, urothelial cancer, biliary tract cancer, glioblastoma, cervical cancer , mesothelioma and colorectal cancer. An isolated immunogenic WT1 peptide comprising an amino acid sequence selected from the group consisting of GAQYRIHTHGVFRGI (SEQ ID NO: 181), LLAAILDFLLLQDPA (SEQ ID NO: 82) and CMTWNQMNLGATLKG (SEQ ID NO: 87) and Each variant has up to three amino acid substitutions, additions or deletions.

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